Bifunctional cytotoxic agents containing the CTI pharmacophore

ABSTRACT

The present invention is directed to novel bifunctional CTI-CTI and CBI-CTI dimers of the formula:
 
F 1 -L 1 -T-L 2 -F 2  
 
where F 1 , L 1 , T, L 2  and F 2  are as defined herein, useful for the treatment for proliferative diseases, where the inventive dimers can function as stand-alone drugs, payloads in antibody-drug-conjugates (ADCs), and linker-payload compounds useful in connection with the production or administration of such ADCs; and to compositions including the aforementioned dimers, linker-payloads and ADCs, and methods for using these dimers, linker-payloads and ADCs, to treat pathological conditions including cancer.

FIELD OF THE INVENTION

The present invention is directed to novel bifunctional CTI-CTI andCBI-CTI dimers useful for the treatment for proliferative diseases. Theinventive dimers can function as stand-alone drugs, payloads inantibody-drug-conjugates (ADCs), and linker-payload compounds useful inconnection with the production or administration of such ADCs. Thepresent invention further relates to compositions including theaforementioned dimers, linker-payloads and ADCs, and methods for usingthese dimers, linker-payloads and ADCs, to treat pathological conditionsincluding cancer.

BACKGROUND

Bifunctional analogs that contain two active DNA alkylation motifs (i.e.a CBI or a CPI) contain two alkylation (e.g., two CPI motifs) fusedtogether. Due to the presence of two reactive alkylation motifs thesecompounds are active DNA cross linkers, whereas compounds with only onealkylation motif (e.g., duocarmycins) are DNA mono-alkylators.

The compounds shown above are representative examples from theliterature and are reported to be potent cytotoxins: A (“GlycosidicProdrugs of Highly Potent Bifunctional Duocarmycin Derivatives forSelective Treatment of Cancer”, Angew. Chem. Int. Ed. 2010, 49,7336-7339; “Duocarmycin Analogues Target Aldehyde Dehydrogenase 1 inLungCancer Cells”, Angew. Chem. Int. Ed. 2012, 51, 2874-2877;“Bifunctional prodrugs and drugs”, WO 2011/054837, DE 10 2009 051 799;“The Two Faces of Potent Antitumor Duocarmycin-Based Drugs: A StructuralDissection Reveals Disparate Motifs for DNA versus AldehydeDehydrogenase 1 Affinity”, Angew. Chem. Int. Ed. 2013, 52, 1-6; B(“Interstrand DNA Cross-linking with Dimers of the SpirocyclopropylAlkylating Moiety of CC-1065”, J. Am. Chem. SOC. 1989, 11 1, 6428-6429;“CC-1065 analogs having two CPI subunits useful as antitumor agents andultraviolet light absorbers”, Eur. Pat. Appl. (1990), EP 359454, alsofor compounds C and D; C (“Synthesis and DNA Cross-Linking by a RigidCPI Dimer”, J. Am. Chem. SOC. 1991, 113, 8994-8995; “NucleotidePreferences for DNA Interstrand Cross-Linking Induced by theCyclopropylpyrroloindole Analogue U-77,779”, Biochemistry 1993, 32,2592-2600; “Determination of the Structural Role of the InternalGuanine-Cytosine Base Pair in Recognition of a Seven-Base-Pair SequenceCross-Linked by Bizelesin”, Biochemistry 1995, 34, 11005-11016;“Analysis of the Monoalkylation and Cross-Linking Sequence Specificityof Bizelesin, a Bifunctional Alkylation AgentRelated to (+)-CC-1065”, J.Am. Chem. SOC. 1993, 115, 5925-5933; “Mapping of DNA Alkylation SitesInduced by Adozelesin and Bizelesin in Human Cells by Ligation-MediatedPolymerase Chain Reaction”, Biochemistry 1994, 33, 6024-6030; “DNAInterstrand Cross-Links Induced by the CyclopropylpyrroloindoleAntitumor Agent Bizelesin Are Reversible upon Exposure to Alkali”,Biochemistry 1993, 32, 9108-9114; “Replacement of the Bizelesin UreadiylLinkage by a Guanidinium Moiety Retards Translocation fromMonoalkylation to Cross-Linking Sites on DNA”, J. Am. Chem. Soc. 1997,119, 3434-3442; “DNA interstrand cross-linking, DNA sequencespecificity, and induced conformational changes produced by a dimericanalog of (+)-CC-1065”, Anti-Cancer Drug Design (1991), 6, 427-452; “Aphase I study of bizelesin, a highly potent and selective DNAinteractiveagent, in patients with advanced solid malignancies”, Ann Oncol. 2003May; 14(5):775-782; “A Phase I study of bizelesin (NSC 615291) inpatients with advanced solid tumors”, Clin Cancer Res. 2002, 3, 712-717;“Solution conformation of a bizelesin A-tract duplex adduct: DNA-DNAcross-linking of an A-tract straightens out bent DNA”, J Mol Biol. 1995,252, 86-101; “Preclinical pharmacology of bizelesin, a potentbifunctional analog of the DNA-binding antibiotic CC-1065”, CancerChemother Pharmacol. 1994, 34, 317-322; and D (“CC-1065 analogs havingtwo CPI subunits useful as antitumor agents and ultraviolet lightabsorbers”, Eur. Pat. Appl. (1990), EP 359454. The active DNA alkylationmotif can in principle exist in either a prodrug form that converts tothe active drug in the biological medium, or in its active state whichdoes not require further conversion. The prodrug-to-active drugconversion for the bifunctional cross linkers is exemplified in the CBIdimer shown below:

A corresponding conversion takes place for all bifunctional crosslinkers that exist in their prodrug states.

Other related bifunctional cross linkers have been reported. (“Chemicaland Biological Explorations of the Family of CC-1065 and the DuocarmycinNatural Products”, Current Topics in Medicinal Chemistry, 2009, 9,1494-1524; “DNA interstrand cross-linking agents and theirchemotherapeutic potential”, Curr Med Chem. 2012, 19, 364-385; “Designand Synthesis of a Novel DNA-DNA Interstrand Adenine-GuanineCross-Linking Agent”, J. Am. Chem. Soc. 2001, 123, 4865-4866; “Effect ofbase sequence on the DNA cross-linking properties ofpyrrolobenzodiazepine (PBD) dimers”, Nucleic Acids Res. 2011, 39,5800-5812; “Sequence-selective recognition of duplex DNA throughcovalent interstrand cross-linking: kinetic and molecular modelingstudies with pyrrolobenzodiazepine dimers”, Biochemistry. 2003, 42,8232-8239; “Bifunctional alkylating agents derived from duocarmycin SA:potent antitumor activity with altered sequence selectivity”, Bioorg MedChem Lett. 2000, 10, 495-498; “Design, Synthesis and CytotoxicityEvaluation of 1-Chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole(seco-CBI) Dimers”, Bioorganic & Medicinal Chemistry 2000, 8, 1607-1617.

A phosphate pro-drug strategy for monomeric seco-CBI containingcytotoxins has been described by Zhao et al. (“Synthesis and biologicalevaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNAalkylators for the targeted treatment of cancer”, J. Med.

Chem. 2012, 55, 766-782) and Zhang et al. (“Immunoconjugates containingphosphate-prodrugged DNA minor groove binding agents, compositionscontaining them, and methods of making them and their use for treatingcancer”, WO 2012/162482).

Certain CBI dimers have recently been described as being useful as ADCPayloads (“I-(Chloromethyl)-2, 3-Dihydro-IH-Benzo[e]indole DimerAntibody-Drug Conjugate Compounds, and Methods of Use and Treatment”,WO2015/023355).

Conjugation of drugs to antibodies, either directly or via linkers,involves a consideration of a variety of factors, including the identityand location of the chemical group for conjugation of the drug, themechanism of drug release, the structural elements providing drugrelease, and the structural modification to the released free drug. Inaddition, if the drug is to be released after antibody internalization,the mechanism of drug release must be consonant with the intracellulartrafficking of the conjugate.

While a number of different drug classes have been tried for delivery byantibodies, only a few drug classes have proved efficacious as antibodydrug conjugates while maintaining a suitable toxicity profile. One suchclass is the auristatins, derivatives of the natural product dolastatin10. Representative auristatins include(N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine) and(N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine). Otherrelated tubulin binding agents include the maytansines (for instance see“Cell-binding agent-maytansinoid conjugates linked via a noncleavablelinker, preparation methods, and methods using them for targetingspecific cell populations” published as WO 2005/037992). Other cytotoxicdrugs that have been employed in linkage with antibodies includeDNA-binding drugs such as calicheamicin that causes sequence-specificdouble-stranded DNA cleavage. Another class of DNA binding cytotoxicdrugs employed in ADCs includes dimeric pyrrolobenzodiazepines (forinstance see “Preparation of unsymmetrical pyrrolobenzodiazepines dimersfor inclusion in targeted conjugates” published as WO2013/041606).Another such class of drug where antibody delivery has been attempted isDNA binding alkylating agents, such as the duocarmycin analog CC-1065(see “Preparation of CC-1065 analogs and their conjugates for treatmentof cancer” published as WO2010/062171) and related compounds (see“Antibody-drug peptide conjugates for use as cytotoxins in cancertreatment” published as WO 2007/038658, and “Immunoconjugates containingphosphate-prodrugged DNA minor groove binding agents, compositionscontaining them, and methods of making them and their use for treatingcancer” published as WO2012/162482). However, these drugs all havelimitations relating to disease indications and treatment profile, andthus there remains a need for additional drugs with improved propertiesdeliverable via antibody conjugation. Accordingly, the present inventionprovides novel ADCs with dimers as payloads.

Another heterocycle that is known to be a potent DNA alkylation motif isrepresented by the “CTI” group(CTI:1,2,8,8a-tetrahydro-4H-cyclopropa[c]thieno[3,2-e]indol-4-one,“Fundamental relationships between structure, reactivity, and biologicalactivity for the duocarmycins and CC-1065”, J. Med. Chem. 2009, 52(19),5771-5780, “Rational Design, Synthesis, and Evaluation of Key Analoguesof CC-1065 and the Duocarmycins”, J. AM. CHEM. SOC. 2007, 129,14092-14099). CTI containing duocarmycin monomers have been described asADC payloads (“Synthesis of functionalized thieno-indole derivs.optionally containing a peptidic residue for the treatment of cancer andtheir use in the preparation of conjugates”, WO 2013/149946,“Preparation of new functionalized alkylating agents containing athieno-indole moiety linked to a DNA-binding moiety for treating cancersand their use in the prepn. of conjugates”, WO 2013/149948). Thestructural differences between “CBI”, “CPI” and “CTI” are depicted inthe following drawing:

CBI, CPI and CTI's differ in the first aromatic ring with the CBI havinga phenyl, the CPI a pyrrole and the CTI a thiophene heterocycle,respectively. The drawing also shows the chloride prodrugs. Theseprodrugs convert into the active drug in the biological medium underloss of hydrogen chloride. Therefore, both the chloride prodrug and theactive cyclopropyl species need to be regarded as equivalent withrespect to their biological activity. The concept of the chlorideprodrugs for CBI, CPI and related groups has been well documented in theliterature (“Design, Synthesis, and Evaluation of Duocarmycin O-AminoPhenol Prodrugs Subject”, J. Med. Chem. 2010, 53, 7731-7738, andreference 8 therein.)

With respect to the CTI group, two structural variations are ofparticular interest, namely the Me-CTI and iso-Me-CTI groups shown inthe following drawing:

Both Me-CTI and iso-Me-CTI groups have been described in context ofduocarmycins (“Chemical and biological explorations of the family ofCC-1065 and the duocarmycin natural products”, Curr Top Med Chem. 2009,9(16), 1494-1524).

SUMMARY OF THE INVENTION

The invention describes new structural dimer analogs based on the CTImotif. The invention also describes spacer elements for thecorresponding CTI dimers and CBI-CTI mixed structures. The term CBI andCTI are used for both the chloride prodrug version as well as the activecyclopropyl species. In addition, linker substitution to these dimericspecies are described as well as the preparation of antibody drugconjugates.

No dimers containing the CTI motif are known and the use of CTI dimersin the present invention results in significant changes in chemicalreactivity and biological properties as compared to previous describeddimer species. Hence, dimers prepared with CTI species representdistinctive drug entities. In this invention, we describe new dimersthat contain either two CTI groups or hybrids that bear one CBI and oneCTI group. Furthermore, we disclose how these dimer species areconnected to suitable linker molecules for the attachment to antibodies.

More specifically, the present invention is directed to cytotoxic dimerscomprising CTI/CTI-based and/or CTI/CBI-based (including seco forms ofCBI and CTI, as detailed herein) dimers, to antibody drug conjugatescomprising such dimers, and to methods for using the same to treatcancer and other indications. Both CTI and CBI structures can berepresented by their seco form and can be substituted and derivatized asdetailed herein. In addition, the phenol function in seco-forms can bederivatized with acetate groups. The phenolic acetate functions arefunctionally equivalent to the phenol as the acetate groups easilyhydrolize to give the free phenols in biological medium.

Thus, the present invention relates to compounds and pharmaceuticalcompositions containing them, to their preparation, and to uses for thecompounds, primarily but not exclusively anti-cancer agents. Accordingto one aspect, the present invention relates to “payload” compound ofFormula I:F¹-L¹-T-L²-F²  (Formula I)or a pharmaceutically acceptable salt or solvate thereof, wherein:F¹ and F² are each independently selected from ring systems A, B, C, D,E, F, G and H:

where:at least one of the ring systems A, B, C and D is present;each R is independently selected from the group consisting of H, —C₁-C₂₀alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, halo, deuterium, hydroxyl,alkoxy, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —NO₂, —C₆-C₁₄ aryl and—C₆-C₁₄ heteroaryl, wherein two or more R optionally join to form a ringor rings, and wherein said —C₆-C₁₄ aryl and —C₆-C₁₄ heteroaryl areoptionally substituted with 1 to 5 substituents independently selectedfrom —C₁-C₁₀ alkyl, —C₁-C₁₀ alkoxy, -halo, —C₁-C₁₀ alkylthio,-trifluoromethyl, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —C₁-C₁₀alkyl-N(C₁-C₈ alkyl)₂, —C₁-C₃ alkylthio, —NO₂ or —C₁-C₁₀ heterocyclyl,for each ring system in which R appears;each V¹ is independently a bond, O, N(R) or S, for each ring system inwhich V appears;each V² is independently O, N(R) or S, for each ring system in which V²appears;W¹ and W² are each independently H, —C₅ alkyl, -phenyl, —C(O)OR,—C(O)SR, —C(O)NHN(R)₂ or —C(O)N(R)₂ for each ring system in which W¹ andW² appear;each X is independently —OH, —O-acyl, azido, halo, cyanate, thiocyanate,isocyanate, thioisocyanate, or

for each ring system in which X appears;each Y is independently selected from the group consisting of H, —C₁-C₆alkyl-R^(A), C(O)R^(A), —C(S)R^(A), C(O)OR^(A), —S(O)₂OR^(A),—C(O)N(R^(A))₂, —C(S)N(R^(A))₂, a carbohydrate, glycosyl, —NO₂,—PO(OR^(A))₂, an amino acid, and a peptide (for instance a peptide thatis cleaved by proteases such as cathepsins and matrixmetalloproteinases) for each ring system in which Y appears, whereineach R^(A) is independently selected from the group consisting of H,—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl and —C₁-C₂₀ alkylN(R)₂, wherein said—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl and —C₁-C₂₀ alkylN(R)₂ are optionallysubstituted with 1 to 3 substitutents independently selected from R;each Z is independently selected from the group consisting of H, —C₁-C₈alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl,—C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂, and —C(O)-halo, and wherein said C₁-C₈ alkyl, —C₁-C₈heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo are each optionally substituted with 1 to 3substitutents independently selected from R, for each ring system inwhich Z appears;L¹ and L² are each independently selected from a direct bond, carbonyl,or a carbonyl acyl group bonded to F¹ or F² at the acyl moiety, wherethe carbonyl acyl group is selected from the group consisting of:

whereinU¹ is selected from H, —CH₃, —OH, —OCH₃, —NO₂, —NH₂, —NHNHAc,—NHNHC(O)CH₃, —NHC(O)phenyl or -halo,U² is H, —OH or —OCH₃,U³ is H, —CH₃ or —C₂H₅,U⁴ is H or CH₃S—,U⁵ and U⁶ are each independently selected from H, -halo, —C₁-C₄ alkyl,—C₁-C₃ alkoxy, —C₁-C₆ dialkylamino, —NO₂, —NHC(O)C₁-C₁₀ alkyl, —OH,—NH₂, —NHC(O)NH₂, —NHC(O)CH₃ or —NHC(O)phenyl,Q¹ is —O—, —S—, or —NH—, andQ² and Q³ are each independently —CH— or —N—;T is selected from:—NHC(O)—,—C(O)NH—,—C(O)O—,—OC(O)—,—NR^(B)-T¹-NR^(C)— where R^(B) and R^(C) are each independently H or—C₁-C₈ alkyl, or together R^(B) and R^(C) join to form a ring andtogether are (CH₂)₂₋₃, where T¹ is selected from the group consisting of—C(O)—, —C(O)(CH₂)_(n)C(O)— where n is an integer from 0 to 50 and—C(O)PhC(O)— where Ph is 1,3- or 1,4-phenylene, and where T¹ isoptionally substituted with 1-2 R,—C(O)hetC(O)— wherein het is a mono-, bi-, or tricyclic heteroaryl of 5to 12 members, containing one, two, or three heteroatoms independentlyselected from O, N, S, P and B, wherein het is optionally substitutedwith 1 to 8 substituents each independently selected from the groupconsisting of —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl,—C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclycl, —NH₂, —NHR^(D) and —NO₂, andsaid optional substituents on het are optionally substituted with R^(E),wherein each R^(D) is independently selected from the group consistingof H, —C₁-C₈ alkyl, —C(O)—C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl,-aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl,—C(O)N(C₁-C₈ alkyl)₂, and —C(O)-halo, optionally substituted with R^(E),wherein each R^(E) is independently selected from the group consistingof H, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈alkyl)₂, and —C(O)-halo, and wherein each R^(E) is optionallysubstituted with 1 to 3 substitutents independently selected from R,—C(A¹)X¹-T²-X¹C(B¹)—, where T² is:

wherein each X¹ is independently a bond, —NR^(E)—, —O— or —S—, whereinA¹ and B¹ are each independently ═O or ═S, wherein R¹, R², R³, and R⁴are each independently R^(E) or R¹ and R² form a ring system, or R³ andR⁴ form a ring system, or both R¹ and R², and R³ and R⁴, eachindependently form ring systems, or R¹ and R³ form a ring system, or R²and R⁴ form a ring system, or both R¹ and R³, and R² and R⁴, eachindependently form ring systems,where said ring systems are independently selected from —C₁-C₁₀heterocyclyl or —C₃-C₈ carbocyclycl, or R¹, R², R³ and R⁴ are each bondsto different carbons on D, wherein g and j are each independently aninteger from 0 to 50 and m is an integer from 1 to 50, and wherein D isa bond or is selected from the group consisting of —S—, —C₁-C₈alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₈heteroalkylene-, -aralkylene, —C₁-C₁₀ heterocyclo and —C₃-C₈ carbocyclo,where said —C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-,—C₁-C₈ heteroalkylene-, -aralkylene, —C₁-C₁₀ heterocyclo and —C₃-C₈carbocyclo are optionally substituted with —R^(E), —C(O)R^(E),—(O)OR^(E), —N(R^(E))₂, —N(R)C(O)R^(E) or —N(R)C(O)OR^(E), and isadditionally optionally substituted by 1 to 2 R, and-G¹-T²-G²-, where G¹ and G² are each independently —S(O)X¹— or—S(O)₂X¹—.

In embodiments of the invention variable n is 0 to 50, preferably 0 to25, preferably 0 to 10, and preferably 1-5. Preferably, variable n maybe 0, 1, 2, 3, 4 or 5.

In other embodiments of the invention the variable —Y— is C(O)N(R^(A))₂or C(S)N(R^(A))₂ where one R^(A) is hydrogen or —C₁-C₂₀ alkyl and theother R^(A) is —C₁-C₂₀ alkyl-N(R)₂, such that the structure:

is formed, where A is oxygen or sulphur.

As noted above, embodiments of the present invention include those whereR¹, R², R³ and R⁴ are each bonds to different carbons on D. When D is a6-membered carbocyclic ring (bold, below), this embodiment may take theform of a cubane:

Other forms of cubanes (for instance substituted forms as outlinedherein) and non-cubanes are also possible and included within theinvention.

According to another aspect of the invention there is provided a“linker-payload” compound of Formula IIA:L-P  (Formula IIA)or a pharmaceutically acceptable salt or solvate thereof, wherein:P is:F¹-L¹-T-L²-F²wherein:F¹ and F² are each independently selected from ring systems A, B, C, D,E, F, G and H:

where:at least one of the ring systems A, B, C and D is present;each R is independently selected from the group consisting of H, —C₁-C₂₀alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, halo, deuterium, hydroxyl,alkoxy, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —NO₂, —C₆-C₁₄ aryl and—C₆-C₁₄ heteroaryl, wherein two or more R optionally join to form a ringor rings, and wherein said —C₆-C₁₄ aryl and —C₆-C₁₄ heteroaryl areoptionally substituted with 1 to 5 substituents independently selectedfrom —C₁-C₁₀ alkyl, —C₁-C₁₀ alkoxy, -halo, —C₁-C₁₀ alkylthio,-trifluoromethyl, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —C₁-C₁₀alkyl-N(C₁-C₈ alkyl)₂, —C₁-C₃ alkylthio, —NO₂ or —C₁-C₁₀ heterocyclyl,for each ring system in which R appears;each V¹ is independently a bond, 0, N(R) or S, for each ring system inwhich V¹ appears;each V² is independently O, N(R) or S, for each ring system in which V²appears;W¹ and W² are each independently H, —C₁-C₅ alkyl, -phenyl, —C(O)OR,—C(O)SR, —C(O)NHN(R)₂ or —C(O)N(R)₂ for each ring system in which W¹ andW² appear;each X is independently selected from —OH, —O-acyl, azido, halo,cyanate, thiocyanate, isocyanate, thioisocyanate, or

for each ring system in which X appears:each Y is independently selected from a bond, H, —C(O)R^(A), C(S)R^(A),C(O)OR^(A), —S(O)₂OR^(A), —C(O)N(R^(A))₂, —C(S)N(R^(A))₂, a carbohydratesuch as glycosyl, —NO₂, —P(O)(OR^(A))₂, an amino acid and a peptide (inparticular a peptide that is cleaved by proteases such as cathepsins andmatrix metalloproteinases) for each ring system in which Y appears,wherein each R^(A) is independently selected from H, —C₁-C₂₀ alkyl,—C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C₁-C₂₀ alkylN(R)₂, —C₁-C₂₀ alkylene, —C₁-C₈heteroalkylene, —C₆-C₁₄ arylene, aralkylene, —C₁-C₁₀ heterocyclo, —C₃-C₈carbocyclo and —C₁-C₂₀ alkylN(R)—, and R^(F) where said R^(A) isooptionally substituted with 1 to 3 substituents independently selectedfrom R, and wherein one Y is divalent and is bonded to L,

R^(F) is —N(R⁶)QN(R⁵)C(O)— and is bonded to L at the carbonyl adjacentN(R⁵), wherein R⁵ and R⁶ are each independently selected from the groupconsisting of H, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl,-aralkyl, —C₁-C₁₀ heterocyclyl and —C₃-C₈ carbocyclyl, or R⁵ or R⁶ joinswith a substituted carbon on Q to form a —C₁-C₁₀ heterocyclic or —C₆-C₁₄heteroaryl ring, or R⁵ and R⁶ join together to form a —C₁-C₁₀heterocyclic or —C₆-C₁₄ heteroaryl ring system, and where Q is —C₁-C₈alkylene-, —C₁-C₈ heteroalkylene-, —C₆-C₁₄ arylene-, -aralkylene-,—C₁-C₈ heterocyclo- or —C₃-C₈ carbocyclo-, wherein Q, R⁵ and R⁶ are eachindependently optionally substituted with 1 to 3 substituentsindependently selected from R;

each Z is independently selected from the group consisting of H, —C₁-C₈alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl,—C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo, and wherein said C₁-C₈ alkyl, —C₁-C₈heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo are each optionally substituted with 1 to 3substitutents independently selected from R, for each ring system inwhich Z appears;L¹ and L² are each independently selected from a direct bond, carbonyl,or a carbonyl acyl group bonded to F¹ or F² at the acyl moiety, wherethe carbonyl acyl group is selected from the group consisting of:

whereinU¹ is selected from H, —CH₃, —OH, —OCH₃, —NO₂, —NH₂, —NHNHAc,—NHNHC(O)CH₃, —NHC(O)phenyl or -halo,U² is H, —OH or —OCH₃,U³ is H, —CH₃ or —C₂H₅,U⁴ is H or CH₃S—,U⁵ and U⁶ are each independently selected from H, -halo, —C₁-C₄ alkyl,—C₁-C₃ alkoxy, —C₁-C₆ dialkylamino, —NO₂, —NHC(O)C₁-C₁₀ alkyl, —OH,—NH₂, —NHC(O)NH₂, —NHC(O)CH₃ or —NHC(O)phenyl,Q¹ is —O—, —S— or —NH—, andQ² and Q³ are each independently —CH— or —N—;T is selected from:—NHC(O)—,—C(O)NH—,—C(O)O—,—OC(O)—,—NR^(B)-T¹-NR^(C)— where R^(B) and R^(C) are each independently H or—C₁-C₈ alkyl, or together R^(B) and R^(C) join to form a ring andtogether are (CH₂)₂₋₃, where T¹ is selected from the group consisting of—C(O)—, —C(O)(CH₂)_(n)C(O)— where n is an integer from 0 to 50 and—C(O)PhC(O)— where Ph is 1,3- or 1,4-phenylene, and where T¹ isoptionally substituted with 1-2 R,—C(O)hetC(O)— wherein het is a mono-, bi-, or tricyclic heteroaryl of 5to 12 members, containing one, two, or three heteroatoms independentlyselected from O, N, S, P and B, where het is optionally substituted with1 to 8 substituents each independently selected from the groupconsisting of —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl,—C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclycl, —NH₂, —NHR^(D) and —NO₂, andsaid optional substituents on het are optionally substituted with R^(E),wherein each R^(D) is independently selected from the group consistingof H, —C₁-C₈ alkyl, —C(O)—C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl,-aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl,—C(O)N(C₁-C₈ alkyl)₂, and —C(O)-halo, optionally substituted with R^(E),wherein each R^(E) is independently selected from the group consistingof H, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, -aryl, -aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈alkyl)₂, and —C(O)-halo, and wherein each R^(E) is optionallysubstituted with 1 to 3 substitutents independently selected from R,—C(A′)X¹-T²-X¹C(B¹)—, where T² is:

wherein each X¹ is independently a bond, —NR^(E)—, —O— or —S—, whereinA¹ and B¹ are each independently ═O or ═S, wherein R¹, R², R³, and R⁴are each independently R^(E) or R¹ and R² form a ring system, or R³ andR⁴ form a ring system, or both R¹ and R², and R³ and R⁴, eachindependently form ring systems, or R¹ and R³ form a ring system, or R²and R⁴ form a ring system, or both R¹ and R³, and R² and R⁴, eachindependently form ring systems, where said ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₈ carbocyclycl,or R¹, R², R³ and R⁴ are each bonds to different carbons on D, wherein gand j are each independently an integer from 0 to 50 and m is an integerfrom 1 to 50, and wherein D is a bond or is selected from the groupconsisting of —S—, —C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄heteroarylene-, —C₁-C₈ heteroalkylene-, -aralkylene, —C₁-C₁₀ heterocycloand —C₃-C₈ carbocyclo, where said —C₁-C₈ alkylene-, —C₆-C₁₄ arylene-,—C₆-C₁₄ heteroarylene-, —C₁-C₈ heteroalkylene-, -aralkylene, —C₁-C₁₀heterocyclo and —C₃-C₈ carbocyclo are optionally substituted with—R^(E), —C(O)R^(E), C(O)OR^(E), —N(R^(E))₂, —N(R)C(O)R^(E) or—N(R)C(O)OR^(E), and is additionally optionally substituted by 1 to 2 R,and-G¹-T²-G²-, where G¹ and G² are each independently —S(O)X¹— or—S(O)₂X′—;L is L^(A)-L^(B)-(L^(C))₁₋₃, wherein L^(A) is selected from the groupconsisting of -halo, —N(R)₂, —CON(R)₂, —S-aryl optionally substitutedwith —NO₂ or —CON(R)₂, —S-heteroaryl optionally substituted with —NO₂,alkyl-SO₂-heteroaryl, arylSO₂-heteroaryl-,

L^(B) is L^(B1)-L^(B2)-L^(B3) wherein L^(B1) is absent or is one or morecomponents selected from the group consisting of —C(O)—, —C(S)—,—C(O)NR—, —C(O)C₁-C₆alkyl-, —C(O)NRC₁-C₆alkyl-,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C(O)C₁-C₆alkylNRC(O)—,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆—C(O)—,—C₁-C₆alkyl-S—S—C₁-C₆alkylNRC(O)CH₂—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆NRC(O)CH₂—,—C(O)C₁-C₆alkyl-NRC(O)C₁₋₆alkyl-, —N═CR-phenyl-O—C₁-C₆alkyl-,—N═CR-phenyl-O—C₁-C₆alkyl-C(O)—, —C(O)—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NRC(O)—,—C(O)C₁-C₆alkyl-phenyl(NR—C(O)C₁-C₆alkyl)₁₋₄-,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—NRC(O)C₁-C₆alkyl-, —C₁-C₆alkyl-, —S—,—C(O)—CH(NR—C(O)C₁-C₆alkyl)-C₁-C₆alkyl- and (—CH₂—CH₂—O—)₁₋₂₀,wherein L^(B2) is AA₀₋₁₂, wherein AA is a natural amino acid, anon-natural amino acid or —(CR¹⁵)_(o)—S—S—(CR¹⁵)_(p) where o and p areeach independently an integer from 1 to 20,L^(B3) is —PABA-, —PABC—, —C(O)(CH₂)_(n)C(O)— or absent;L^(C) is absent or independently selected from the group consisting of—C₁-C₆alkylene-, —NRC₃-C₈-heterocyclylNR—, —NRC₃-C₈-carbocyclylNR—,—NRC₁-C₆alkylNR—, —NRC₁-C₆alkylene-, —S—, —NR—, —NRNR—,—O(CR₂)₁₋₄S—S(CR₂)₁₋₄N(R)—, —NRC₁-C₆-alkylenephenyleneNR—,—NRC₁-C₆alkylenephenyleneSO₂NR—, —OC₁-C₆alkylS-SC₁-C₆alkylC(COOR)NR—,—NRC(COOR)C₁-C₆alkylS-SC₁-C₆alkylO—,

whereinX^(A) is CR or N,X^(B) is CH, CR(C(R)₂)₁₋₃NR, CR(C(R)₂)₁₋₃O, CR(C(R)₂)₁₋₃C(O)NR,CR—(C(R)₂)₁₋₃C(O)NRNR, CR(C(R)₂)₁₋₃SO₂NR, CR(C(R)₂)₁₋₃NRNR,CR(C(R)₂)₁₋₃NRC(O) or N,each X^(C) is R,each X^(D) is —(CH₂)₁₋₅—, or is absent;X^(E) is O, S, (R)₂, C(R)(C(R)₂)₁₋₃—NR₂ or NR andeach X^(F) is (C(R)₂)₁₋₃—NR or C(R)₂—(C(R)₂)₁₋₃—O.

In other embodiments of the invention the variable —Y— is C(O)N(R^(A))₂or C(S)N(R^(A))₂ where one R^(A) is hydrogen or C₁-C₂₀ alkyl and theother R^(A) is C₁-C₂₀ alkyl-N(R)—, such that the structure:

is formed, where each A is independently oxygen or sulphur.

According to still another aspect of the invention there is provided anantibody drug conjugate compound of Formula IIIA:AB-(L-P)₁₋₂₀  (Formula IIIA)or a pharmaceutically acceptable salt or solvate thereof, wherein:AB is an antibody;P is:F¹-L¹-T-L²-F²wherein:F¹ and F² are each independently selected from ring systems A, B, C, D,E, F, G and H:

where:at least one of the ring systems A, B, C and D is present;each R is independently selected from the group consisting of H, —C₁-C₂₀alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, halo, deuterium, hydroxyl,alkoxy, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —NO₂, —C₆-C₁₄ aryl and—C₆-C₁₄ heteroaryl, wherein two or more R optionally join to form a ringor rings, and wherein said —C₆-C₁₄ aryl and —C₆-C₁₄ heteroaryl areoptionally substituted with 1 to 5 substituents independently selectedfrom —C₁-C₁₀ alkyl, —C₁-C₁₀ alkoxy, -halo, —C₁-C₁₀ alkylthio,-trifluoromethyl, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —C₁-C₁₀alkyl-N(C₁-C₈ alkyl)₂, —C₁-C₃ alkylthio, —NO₂ or —C₁-C₁₀ heterocyclyl,for each ring system in which R appears;each V¹ is independently a bond, O, N(R) or S, for each ring system inwhich V¹ appears;each V² is independently O, N(R) or S, for each ring system in which V²appears;W¹ and W² are each independently H, —C₁-C₅ alkyl, -phenyl, —C(O)OR,—C(O)SR, —C(O)NHN(R)₂ or —C(O)N(R)₂ for each ring system in which W¹ andW² appear;each X is independently selected from —OH, —O-acyl, azido, halo,cyanate, thiocyanate, isocyanate, thioisocyanate, or

for each ring system in which X appears;each Y is independently selected from a bond, H, —C(O)R^(A), C(S)R^(A),C(O)OR^(A), —S(O)₂R^(A), —C(O)N(R^(A))₂, —C(S)N(R^(A))₂, a carbohydratesuch as glycosyl, —NO₂, —P(O)(OR^(A))₂, an amino acid, and a peptide (inparticular a peptide that is cleaved by proteases such as cathepsins andmatrix metalloproteinases) for each ring system in which Y appears,wherein each R^(A) is independently selected from H, —C₁-C₂₀ alkyl,—C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C₁-C₂₀ alkylN(R)₂, —C₁-C₂₀ alkylene, —C₁-C₈heteroalkylene, —C₆-C₁₄ arylene, aralkylene, —C₁-C₁₀ heterocyclo, —C₃-C₈carbocyclo and —C₁-C₂₀ alkylN(R)—, and R^(F) where said R^(A) isoptionally substituted with 1 to 3 substituents independently selectedfrom R, and wherein one Y is divalent and is bonded to L,R^(F) is —N(R⁶)QN(R⁵)C(O)— and is bonded to L at the carbonyl adjacentN(R⁵), wherein R⁵ and R⁶ are each independently selected from the groupconsisting of H, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl,-aralkyl, —C₁-C₁₀ heterocyclyl and —C₃-C₈ carbocyclyl, or R⁵ or R⁶ joinswith a substituted carbon on Q to form a —C₁-C₁₀ heterocyclic or —C₆-C₁₄heteroaryl ring, or R⁵ and R⁶ join together to form a —C₁-C₁₀heterocyclic or —C₆-C₁₄ heteroaryl ring system, and where Q is —C₁-C₈alkylene-, —C₁-C₈ heteroalkylene-, —C₆-C₁₄ arylene-, -aralkylene-,—C₁-C₁₀ heterocyclo- or —C₃-C₈ carbocyclo-, wherein Q, R⁵ and R⁶ areeach independently optionally substituted with 1 to 3 substituentsindependently selected from R;each Z is independently selected from the group consisting of H, —C₁-C₈alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl,—C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo, and wherein said C₁-C₈ alkyl, —C₁-C₈heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo are each optionally substituted with 1 to 3substitutents independently selected from R, for each ring system inwhich Z appears;L¹ and L² are each independently selected from a direct bond, carbonyl,or a carbonyl acyl group bonded to F¹ or F² at the acyl moiety, wherethe carbonyl acyl group is selected from the group consisting of:

whereinU¹ is selected from H, —CH₃, —OH, —OCH₃, —NO₂, —NH₂, —NHNHAc,—NHNHC(O)CH₃, —NHC(O)phenyl or -halo,U² is H, —OH or —OCH₃,U³ is H, —CH₃ or —C₂H₅,U⁴ is H or CH₃S—,U⁵ and U⁶ are each independently selected from H, -halo, —C₁-C₄ alkyl,—C₁-C₃ alkoxy, —C₁-C₆ dialkylamino, —NO₂, —NHC(O)C₁-C₁₀ alkyl, —OH,—NH₂, —NHC(O)NH₂, —NHC(O)CH₃ or —NHC(O)phenyl,Q¹ is —O—, —S— or —NH—,Q² and Q³ are each independently —CH— or —N—;T is selected from:—NHC(O)—,—C(O)NH—,—C(O)O—,—OC(O)—,—NR^(B)-T-NR^(C)— where R^(B) and R^(C) are each independently H or—C₁-C₈ alkyl, or together R^(B) and R^(C) join to form a ring andtogether are (CH₂)₂₋₃, where T¹ is selected from the group consisting of—C(O)—, —C(O)(CH₂)_(n)C(O)— where n is an integer from 0 to 50 and—C(O)PhC(O)— where Ph is 1,3- or 1,4-phenylene, and where T¹ isoptionally substituted with 1-2 R,—C(O)hetC(O)— wherein het is a mono-, bi-, or tricyclic heteroaryl of 5to 12 members, containing one, two, or three heteroatoms independentlyselected from O, N, S, P and B, where het is optionally substituted with1 to 8 substituents each independently selected from the groupconsisting of —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl,—C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclycl, —NH₂, —NHR^(D) and —NO₂, andsaid optional substituents on het are optionally substituted with R^(E),wherein each R^(D) is independently selected from the group consistingof H or —C₁-C₈ alkyl, —C(O)—C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄aryl, -aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈alkyl, —C(O)N(C₁-C₈ alkyl)₂, and —C(O)-halo, optionally substituted withR^(E),wherein each R^(E) is independently selected from the group consistingof H, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, -aryl, -aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈alkyl)₂, and —C(O)-halo, and wherein each R^(E) is optionallysubstituted with 1 to 3 substitutents independently selected from R,—C(A¹)X¹-T²-X¹C(B¹)—, where T² is:

wherein each X¹ is independently a bond, —NR^(E)—, —O— or —S—, whereinA¹ and B¹ are each independently ═O or ═S, wherein R¹, R², R³, and R⁴are each independently R^(E) or R¹ and R² form a ring system, or R³ andR⁴ form a ring system, or both R¹ and R², and R³ and R⁴, eachindependently form ring systems, or R and R³ form a ring system, or R²and R⁴ form a ring system, or both R¹ and R³, and R² and R⁴, eachindependently form ring systems, where said ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₈ carbocyclycl,or R⁴, R², R³ and R⁴ are each bonds to different carbons on D, wherein gand j are each independently an integer from 0 to 50 and m is an integerfrom 1 to 50, and wherein D is a bond or is selected from the groupconsisting of —S—, —C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄heteroarylene-, —C₁-C₈ heteroalkylene-, -aralkylene, —C₁-C₁₀ heterocycloand —C₃-C₈ carbocyclo, where said —C₁-C₈ alkylene-, —C₆-C₁₄ arylene-,—C₆-C₁₄ heteroarylene-, —C₁-C₈ heteroalkylene-, -aralkylene, —C₁-C₁₀heterocyclo and —C₃-C₈ carbocyclo are optionally substituted with—R^(E), —C(O)R^(E), C(O)OR^(E), —N(R^(E))₂, —N(R)C(O)R^(E) or—N(R)C(O)OR^(E), and D is additionally optionally substituted by 1 to 2R, and-G¹-T²-G²-, where G and G² are each independently —S(O)X¹— or —S(O)₂X⁴—;L is L^(A)-L^(B)-(L^(C))₁₋₃;L^(A) is selected from: a bond to AB, —NR-(bond to AB),-heteroaryl-(bond to AB),

L^(B) is L^(B1)-L^(B2)-L^(B3)wherein L^(B1) is absent or is one or more components selected from thegroup consisting of —C(O)—, —C(S)—, —C(O)NR—, —C(O)C₁-C₆alkyl-,—C(O)NRC₁-C₆alkyl-, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C(O)C₁-C₆alkylNRC(O)—,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆—C(O)—,—C₁-C₆alkyl-S—S—C₁-C₆alkylNRC(O)CH₂—,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NR^(C)(O)CH₂—,—C(O)C₁-C₆alkyl-NR^(C)(O)C₁₋₆alkyl-, —N═CR-phenyl-O—C₁-C₆alkyl-,—N═CR-phenyl-O—C₁-C₆alkyl-C(O)—, —C(O)—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NR^(C)(O)—,—C(O)C₁-C₆alkyl-phenyl(NR—C(O)C₁-C₆alkyl)₁₋₄-,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—NR^(C)(O)C₁-C₆alkyl-, —C₁-C₆alkyl-, —S—,—C(O)—CH(NR—C(O)C₁-C₆alkyl)-C₁-C₆alkyl- and (—CH₂—CH₂—O—)₁₋₂₀;L^(B2) is AA₀₋₁₂, wherein AA is a natural amino acid, a non-naturalamino acid or —(CR¹⁵)_(o)—S—S—(CR¹⁵)_(p) where o and p are eachindependently an integer from 1 to 20,L^(B3) is —PABA-, —PABC—, —C(O)(CH₂)_(n)C(O)— or absent;L^(C) is absent or is independently selected from the group consistingof —C₁-C₆alkylene-, —NRC₃-C₈-heterocyclylNR—, —NRC₃-C₈-carbocyclylNR—,—NRC₁-C₆alkylNR—, —NRC₁-C₆alkylene-, —S—, —NR—, —NRNR—,—O(CR₂)₁₋₄S—S(CR₂)₁₋₄N(R)—, —NRC₁-C₆-alkylenephenyleneNR—,—NRC₁-C₆alkylenephenyleneSO₂NR—, —OC₁-C₆alkylS-SC₁-C₆alkylC(COOR)NR—,—NR^(C)(COOR)C₁-C₆alkylS-SC₁-C₆alkylO—,

whereinX^(A) is CR or N,X^(B) is CH, CR(C(R)₂)₁₋₃NR, CR(C(R)₂)₁₋₃O, CR(C(R)₂)₁₋₃C(O)NR,CR—(C(R)₂)₁₋₃C(O)NRNR, CR(C(R)₂)₁₋₃SO₂NR, CR(C(R)₂)₁₋₃NRNR,CR(C(R)₂)₁₋₃NR^(C)(O) or N,each X^(C) is R;each X^(D) is —(CH)₁₋₅—, or is absent;X^(E) is O, S, C(R)₂, C(R)(C(R)₂)₁₋₃—NR₂ or NR, andeach X^(F) is (C(R)₂)₁₋₃—NR or C(R)₂—(C(R)₂)₁₋₃—O.

According to another aspect of the invention there is provided a“linker-payload” compound of Formula IIB:

or a pharmaceutically acceptable salt or solvate thereof, wherein:F¹ and F² are each independently selected from ring systems A, B, C, D,E, F, G and H:

where:at least one of the ring systems A, B, C and D is present;each R is independently selected from the group consisting of H, —C₁-C₂₀alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, halo, deuterium, hydroxyl,alkoxy, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —NO₂, —C₆-C₁₄ aryl and—C₆-C₁₄ heteroaryl, wherein two or more R optionally join to form a ringor rings, and wherein said —C₆-C₁₄ aryl and —C₆-C₁₄ heteroaryl areoptionally substituted with 1 to 5 substituents independently selectedfrom —C₁-C₁₀ alkyl, —C₁-C₁₀ alkoxy, -halo, —C₁-C₁₀ alkylthio,-trifluoromethyl, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —C₁-C₁₀alkyl-N(C₁-C₈ alkyl)₂, —C₁-C₃ alkylthio, —NO₂ or —C₁-C₁₀ heterocyclyl,for each ring system in which R appears;each V¹ is independently a bond, O, N(R) or S, for each ring system inwhich V¹ appears;each V² is independently O, N(R) or S, for each ring system in which V²appears;W¹ and W² are each independently H, —C₁-C₅ alkyl, -phenyl, —C(O)OR,—C(O)SR, —C(O)NHN(R)₂ or —C(O)N(R)₂ for each ring system in which W¹ andW² appear;each X is independently —OH, —O-acyl, azido, halo, cyanate, thiocyanate,isocyanate, thioisocyanate, or

for each ring system in which X appears;each Y is independently selected from the group consisting of H, —C₁-C₆alkyl-R^(A) —C(O)R^(A), —C(S)R^(A), C(O)OR^(A), —S(O)₂OR^(A),—C(O)N(R^(A))₂, —C(S)N(R^(A))₂, a carbohydrate such as glycosyl, —NO₂,—PO(OR^(A))₂, an amino acid and a peptide (in particular a peptide thatis cleaved by proteases such as cathepsins and matrixmetalloproteinases) for each ring system in which Y appears, whereineach R^(A) is independently selected from the group consisting of H,—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl and —C₁-C₂₀ alkylN(R)₂, wherein said—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₀ carbocyclyl and —C₁-C₂₀ alkylN(R)₂ are optionallysubstituted with 1 to 3 substitutents independently selected from R;each Z is independently selected from the group consisting of H, —C₁-C₈alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl,—C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo, and wherein said C₁-C₈ alkyl, —C₁-C₈heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo are each optionally substituted with 1 to 3substitutents independently selected from R, for each ring system inwhich Z appears;L¹ and L² are each independently selected from a direct bond, carbonyl,or a carbonyl acyl group bonded to F¹ or F² at the acyl moiety, wherethe carbonyl acyl group is selected from the group consisting of:

whereinU¹ is selected from H, —CH₃, —OH, —OCH₃, —NO₂, —NH₂, —NHNHAc,—NHNHC(O)CH₃, —NH—C(O)phenyl or -halo,U² is H, —OH or —OCH₃,U³ is H, —CH₃ or —C₂H₅,U⁴ is H or CH₃S—,U⁵ and U⁶ are each independently selected from H, -halo, —C₁-C₄ alkyl,—C₁-C₃ alkoxy, —C₁-C₆ dialkylamino, —NO₂, —NHC(O)C₁-C₁₀ alkyl, —OH,—NH₂, —NHC(O)NH₂, —NHC(O)CH₃ or —NHC(O)phenyl,Q¹ is —O—, —S— or —NH—, andQ² and Q³ are each independently —CH— or —N—;T is selected from:—C(A¹)X¹-T²-X¹C(B¹)—, where T² is:

wherein each X¹ is independently a bond, —NR^(E)—, —O— or —S—, whereinA¹ and B¹ are each independently ═O or ═S, wherein R¹, R², R³, and R⁴are each independently R^(E), or R¹ and R² form a ring system, or R³ andR⁴ form a ring system, or both R¹ and R², and R³ and R⁴ eachindependently form ring systems, or R¹ and R³ form a ring system, or R²and R⁴ form a ring system, or both R¹ and R³, and R² and R⁴ eachindependently form ring systems, where the ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₈ carbocyclycl,or R¹, R², R³ and R⁴ are each bonds to different carbons on D, wherein gand j are each independently an integer from 0 to 50 and m is an integerfrom 1 to 50, and wherein D is selected from the group consisting of—C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₈heteroalkylene-, -aralkylene, —C₁-C₁₀ heterocyclo and —C₃-C₈ carbocyclo,where said —C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-,—C₁-C₈ heteroalkylene-, -aralkylene, —C₁-C₁₀ heterocyclo and —C₃-C₈carbocyclo are substituted with one member of the group selected fromN(R^(E))C(O)— where the carbonyl is bonded to L, and —C(O)— where thecarbonyl is bonded to L, and additionally optionally substituted by 1 to2 R;where each R^(E) is independently selected from the group consisting ofH, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, -aryl, -aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈alkyl)₂, and —C(O)-halo, and wherein each R^(E) is optionallysubstituted with 1 to 3 substitutents independently selected from R;L is L^(A)-L^(B)-(L^(C))₁₋₃;L^(A) is selected from -halo, —N(R)₂, —CON(R)₂, —S-aryl optionallysubstituted with —NO₂ or —CONR₂, —S-heteroaryl optionally substitutedwith —NO₂, alkyl-SO₂-heteroaryl, arylSO₂-heteroaryl-,

L^(B) is L^(B1)-L^(B2)-L^(B3)wherein L^(B1) is absent or is one or more components selected from thegroup consisting of —C(O)—, —C(S)—, —C(O)NR—, —C(O)C₁-C₆alkyl-,—C(O)NRC₁-C₆alkyl-, —C₁-C₆alkyl(OCH₂CH₂)₁₋₅—, —C(O)C₁-C₆alkylNRC(O)—,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₅—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₅—C(O)—,—C₁-C₆alkyl-S—S—C₁-C₆alkylNRC(O)CH₂—,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NR^(C)(O)CH₂—, —C(O)C₁-C₆alkyl-NR^(C)(O)C₁,-alkyl-, —N═CR-phenyl-O—C₁-C₆alkyl-, —N═CR-phenyl-O—C₁-C₆alkyl-C(O)—,—C(O)—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NRC(O)—,—C(O)C₁-C₆alkyl-phenyl(NR—C(O)C₁-C₆alkyl)₁₋₄-,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₅—NR^(C)(O)C₁-C₆alkyl-, —C₁-C₆alkyl-, —S—,—C(O)—CH(NR—C(O)C₁-C₆alkyl)-C₁-C₆alkyl- and (—CH₂—CH₂—O—)₁₋₂₀;L^(B2) is AA₀₋₁₂, wherein AA is a natural amino acid, a non-naturalamino acid or —(CR¹⁵)_(o)—S—S—(CR¹⁵)_(p) where o and p are eachindependently an integer from 1 to 20,L^(B3) is —PABA-, —PABC—, —C(O)(CH₂)_(n)C(O)— or absent;L^(C) is absent or is independently selected from the group consistingof —C₁-C₆alkylene-, —NRC₃-C₈-heterocyclylNR—, —NRC₃-C₈-carbocyclylNR—,—NRC₁-C₆alkylNR—, —NRC₁-C₆alkylene-, —S—, —NR—, —NRNR—,—O(CR₂)₁₋₄S—S(CR₂)₁₋₄N(R)—, —NRC₁-C₅-alkylenephenyleneNR—,—NRC₁-C₆alkylenephenyleneSO₂NR—, —OC₁-C₆alkylS-SC₁-C₆alkylC(COOR)NR—,—NR^(C)(COOR)C₁-C₆alkylS-SC₁-C₆alkylO—,

whereinX^(A) is CR or N,X^(B) is CH, CR(C(R)₂)₁₋₃NR, CR(C(R)₂)₁₋₃O, CR(C(R)₂)₁₋₃C(O)NR,CR—(C(R)₂)₁₋₃C(O)NRNR, CR(C(R)₂)₁₋₃SO₂NR, CR(C(R)₂)₁₋₃NRNR,CR(C(R)₂)₁₋₃NR^(C)(O) or N;each X^(C) is R;each X^(D) is —(CH₂)₁₋₅—, or is absent;X^(E) is O, S, C(R)₂, C(R)(C(R)₂)₁₋₃—NR₂ or NR, andeach X^(F) is (C(R)₂)₁₋₃—NR or C(R)₂—(C(R)₂)₁₋₃—O.

According to yet another aspect of the invention there is provided anantibody drug conjugate compound of Formula IIIB:

or a pharmaceutically acceptable salt or solvate thereof, wherein:AB is an antibody;F¹ and F² are each independently selected from ring systems A, B, C, D,E, F, G and H:

where:at least one of the ring systems A, B, C and D is present;each R is independently selected from the group consisting of H, —C₁-C₂₀alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, halo, deuterium, hydroxyl,alkoxy, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —NO₂, —C₆-C₁₄ aryl and—C₆-C₁₄ heteroaryl, wherein two or more R optionally join to form a ringor rings, and wherein said —C₆-C₁₄ aryl and —C₆-C₁₄ heteroaryl areoptionally substituted with 1 to 5 substituents independently selectedfrom —C₁-C₁₀ alkyl, —C₁-C₁₀ alkoxy, -halo, —C₁-C₁₀ alkylthio,-trifluoromethyl, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —C₁-C₁₀alkyl-N(C₁-C₈ alkyl)₂, —C₁-C₃ alkylthio, —NO₂ or —C₁-C₁₀ heterocyclyl,for each ring system in which R appears;each V¹ is independently a bond, 0, N(R) or S, for each ring system inwhich V¹ appears;each V² is independently O, N(R) or S, for each ring system in which V²appears;W¹ and W² are each independently H, —C₁-C₅ alkyl, -phenyl, —C(O)OR,—C(O)SR, —C(O)NHN(R)₂ or —C(O)N(R)₂ for each ring system in which W¹ andW² appear;each X is independently —OH, —O-acyl, azido, halo, cyanate, thiocyanate,isocyanate, thioisocyanate, or

for each ring system in which X appears;each Y is independently selected from the group consisting of H, —C₁-C₆alkyl-R^(A) —C(O)R^(A), —C(S)R^(A), C(O)OR^(A), —S(O)₂OR^(A),—C(O)N(R^(A))₂, —C(S)N(R^(A))₂, a carbohydrate such as glycosyl, —NO,—PO(OR^(A))₂, an amino acid, and a peptide (in particular a peptide thatis cleaved by proteases such as cathepsins and matrixmetalloproteinases), for each ring system in which Y appears, whereineach R^(A) is independently selected from the group consisting of H,—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl and —C₁-C₂₀ alkylN(R)₂, wherein said—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₀ carbocyclyl and —C₁-C₂₀ alkylN(R)₂ are optionallysubstituted with 1 to 3 substitutents independently selected from R;each Z is independently selected from the group consisting of H, —C₁-C₈alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl,—C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo, and wherein said C₁-C₈ alkyl, —C₁-C₈heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo are each optionally substituted with 1 to 3substitutents independently selected from R, for each ring system inwhich Z appears;L¹ and L² are each independently selected from a direct bond, carbonyl,or a carbonyl acyl group bonded to F¹ or F² at the acyl moiety, wherethe carbonyl acyl group is selected from the group consisting of:

whereinU¹ is selected from H, —CH₃, —OH, —OCH₃, —NO₂, —NH₂, —NHNHAc,—NHNHC(O)CH₃, —NH—C(O)phenyl or -halo,U² is H, —OH or —OCH₃,U³ is H, —CH₃ or —C₂H₅,U⁴ is H or CH₃S—,U⁵ and U⁶ are each independently selected from H, -halo, —C₁-C₄ alkyl,—C₁-C₃ alkoxy, —C₁-C₆ dialkylamino, —NO₂, —NHC(O)C₁-C₁₀ alkyl, —OH,—NH₂, —NHC(O)NH₂, —NHC(O)CH₃ or —NHC(O)phenyl,Q¹ is —O—, —S— or —NH—, andQ² and Q³ are each independently —CH— or —N—;T is selected from:—C(A¹)X¹-T²-X¹C(B¹)—, where T² is:

wherein each X¹ is independently a bond, —NR^(E)—, —O— or —S—, whereinA¹ and B¹ are each independently ═O or ═S, wherein R¹, R², R³, and R⁴are each independently R^(E), or R¹ and R² form a ring system, or R³ andR⁴ form a ring system, or both R¹ and R², and R³ and R⁴ eachindependently form ring systems, or R and R³ form a ring system, or R²and R⁴ form a ring system, or both R¹ and R³, and R² and R⁴ eachindependently form ring systems, where the ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₈ carbocyclycl,or R¹, R², R³ and R⁴ are each bonds to different carbons on D, wherein gand j are each independently an integer from 0 to 50 and m is an integerfrom 1 to 50, and wherein D is selected from the group consisting of—C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₈heteroalkylene-, -aralkylene, —C₁-C₁₀ heterocyclo and —C₃-C₈ carbocyclo,where said —C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-,—C₁-C₈ heteroalkylene-, -aralkylene, —C₁-C₁₀ heterocyclo and —C₃-C₈carbocyclo are substituted with one member of the group selected fromN(R^(E))C(O)— where the carbonyl is bonded to L, and —C(O)— where thecarbonyl is bonded to L, and additionally optionally substituted by 1 to2 R;where each R^(E) is independently selected from the group consisting ofH, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, -aryl, -aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈alkyl)₂, and —C(O)-halo, and wherein each R^(E) is optionallysubstituted with 1 to 3 substitutents independently selected from R;L is L^(A)-L^(B)-(L^(C))₁₋₃;L^(A) is selected from: a bond to AB, —NR-(bond to AB),-heteroaryl-(bond to AB),

L^(B) is L^(B1)-L^(B2)-L^(B3)wherein L^(B1) is absent or is one or more components selected from thegroup consisting of —C(O)—, —C(S)—, —C(O)NR—, —C(O)C₁-C₆alkyl-,—C(O)NRC₁-C₆alkyl-, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C(O)C₁-C₆alkylNRC(O)—,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆—C(O)—,—C₁-C₆alkyl-S—S—C₁-C₆alkylNRC(O)CH₂—,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NR^(C)(O)CH₂—,—C(O)C₁-C₆alkyl-NR^(C)(O)C₁₋₆alkyl-, —N═CR-phenyl-O—C₁-C₆alkyl-,—N═CR-phenyl-O—C₁-C₆alkyl-C(O)—, —C(O)—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NR^(C)(O)—,—C(O)C₁-C₆alkyl-phenyl(NR—C(O)C₁-C₆alkyl)₁₋₄-,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—NR^(C)(O)C₁-C₆alkyl-, —C₁-C₆alkyl-, —S—,—C(O)—CH(NR—C(O)C₁-C₆alkyl)-C₁-C₆alkyl- and (—CH₂—CH₂—O—)₁₋₂₀;L^(B2) is AA₀₋₁₂, wherein AA is a natural amino acid, a non-naturalamino acid or —(CR¹⁵)_(o)—S—S—(CR¹⁵)_(p) where o and p are eachindependently an integer from 1 to 20,L^(B3) is —PABA-, —PABC—, —C(O)(CH₂)_(n)C(O)— or absent;L^(C) is absent or is independently selected from the group consistingof —C₁-C₆alkylene-, —NRC₃-C₈-heterocyclylNR—, —NRC₃-C₈-carbocyclylNR—,—NRC₁-C₆alkylNR—, —NRC₁-C₆alkylene-, —S—, —NR—, —NRNR—,—O(CR₂)₁₋₄S—S(CR₂)₁₋₄N(R)—, —NRC₁-C₆-alkylenephenyleneNR—,—NRC₁-C₆alkylenephenyleneSO₂NR—, —OC₁-C₆alkylS-SC₁-C₆alkylC(COOR)NR—,—NR^(C)(COOR)C₁-C₆alkylS-SC₁-C₆alkylO—,

whereinX^(A) is CR or N,X^(B) is H, CR(C(R)₂)₁₋₃NR, CR(C(R)₂)₁₋₃O, CR(C(R)₂)₁₋₃C(O)NR,CR—(C(R)₂)₁₋₃C(O)NRNR, CR(C(R)₂)₁₋₃SO₂NR, CR(C(R)₂)₁₋₃NRNR,CR(C(R)₂)₁₋₃NRC(O) or N;each X^(C) is R;each X^(D) is —(CH₂)₁₋₅—, or is absent;X^(E) is O, S, C(R)₂, C(R)(C(R)₂)₁₋₃—NR₂ or NR, andeach X^(F) is (C(R)₂)₁₋₃—NR or C(R)₂—(C(R)₂)₁₋₃—O.

Additional aspects of the invention include compounds such as thosementioned herein where

each R is independently selected from the group consisting of H,deuterium, —C₁-C₂₀ alkyl and —NH₂;

each V¹ is independently O or N(R) for each ring system in which V¹appears;

each V² is independently O or N(R) for each ring system in which V²appears;

W and W² are each independently H, —C₁-C₈ alkyl, —C(O)OR, or —C(O)NR₂for each ring system in which W¹ and W² appear;

each X is independently halo, for each ring system in which X appears;

each Y is independently selected from the group consisting of H,—C(O)R^(A), —C(O)N(R^(A))₂, a carbohydrate such as glycosyl, —NO₂,—PO(OR^(A))₂, an amino acid and a peptide (in particular a peptide thatis cleaved by proteases such as cathepsins and matrixmetalloproteinases) for each ring system in which Y appears, whereineach R^(A) is independently selected from the group consisting of H,—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₃-C₈ carbocyclyl and —C₁-C₂₀alkylN(R)₂, wherein said —C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₃-C₈carbocyclyl and —C₁-C₂₀ alkylN(R)₂ are optionally substituted with 1 to3 substitutents independently selected from R;L¹ and L² are each independently selected from a direct bond andcarbonyl; andT is selected from:—NR^(B)-T¹-NR^(C)— where R^(B) and R^(C) are each independently H or—C₁-C₈ alkyl,—C(O)hetC(O)— wherein het is a monocyclic heteroaryl of 5 to 12 members,containing one or two heteroatoms independently selected from O, N andS, wherein het is optionally substituted with 1 to 8 substituents eachindependently selected from the group consisting of —C₁-C₈ alkyl, —NH₂,and —NH₂, and said optional substituents on het are optionallysubstituted with —C₁-C₈ alkyl, and—C(A¹)X¹-T²-X^(C)(B¹)—, where T² is:

wherein each X¹ is a bond, wherein A¹ and B¹ are each independently ═O,wherein R¹, R², R³, and R⁴ are each independently H or R¹ and R² form aring system, or R³ and R⁴ form a ring system, or both R¹ and R², and R³and R⁴, each independently form ring systems, or R¹ and R³ form a ringsystem, or R² and R⁴ form a ring system, or both R¹ and R³, and R² andR⁴, each independently form ring systems, where said ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₀ carbocyclycl,and wherein D is a bond or is selected from the group consisting of —S—,—C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₁₀heterocyclo and —C₃-C₈ carbocyclo, where said —C₁-C₈ alkylene-, —C₆-C₁₄arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₁₀ heterocyclo and —C₃-C₈carbocyclo are optionally substituted with —NH₂, —N(R)C(O)H or—N(R)C(O)OH.

Additional aspects of the invention include compounds such as thosementioned herein where two or more R optionally join to form a ring orrings.

Additional aspects of the invention include compounds such as thosementioned herein where

each R is independently selected from the group consisting of H,deuterium, —C₁-C₂₀ alkyl and —NH₂;

each V¹ is independently O or N(R) for each ring system in which V⁴appears;

each V² is independently O or N(R) for each ring system in which V²appears;

W¹ and W² are each independently H, —C₁-C₅ alkyl, —C(O)OR, or —C(O)NR₂for each ring system in which W¹ and W² appear;

each X is independently halo, for each ring system in which X appears;

each Y is independently selected from a bond, H, —C(O)R^(A), C(S)R^(A),C(O)OR^(A), —S(O)₂R^(A), —C(O)N(R^(A))₂, —C(S)N(R^(A))₂, a carbohydratesuch as glycosyl, —NO₂, —P(O)(OR^(A))₂, an amino acid, and a peptide (inparticular a peptide that is cleaved by proteases such as cathepsins andmatrix metalloproteinases) for each ring system in which Y appears,wherein each R^(A) is independently selected from H, —C₁-C₂₀ alkyl,—C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C₁-C₂₀ alkylN(R)₂, —C₁-C₂₀ alkylene, —C₁-C₈heteroalkylene, —C₆-C₁₄ arylene, aralkylene, —C₁-C₁₀ heterocyclo, —C₃-C₈carbocyclo and —C₁-C₂₀ alkylN(R)—, and R^(F) where said R^(A) isoptionally substituted with 1 to 3 substituents independently selectedfrom R, and wherein one Y is divalent and is bonded to L,R^(F) is —N(R⁶)QN(R⁵)C(O)— and is bonded to L at the carbonyl adjacentN(R⁵), wherein R⁵ and R⁶ are each independently selected from the groupconsisting of H, —C₁-C₈ alkyl, and —C₁-C₈ heteroalkyl, or R⁵ or R⁶ joinswith a substituted carbon on Q to form a —C₁-C₁₀ heterocyclic or —C₆-C₁₄heteroaryl ring, or R⁵ and R⁶ join together to form a —C₁-C₁₀heterocyclic or —C₆-C₁₄ heteroaryl ring system, and where Q is —C₁-C₈alkylene-, —C₆-C₁₄ arylene-, or —C₃-C₈ carbocyclo-, wherein Q, R⁵ and R⁶are each independently optionally substituted with 1 to 3 substituentsindependently selected from R;L¹ and L² are each independently selected from a direct bond andcarbonyl; andT is selected from:—NR^(B)-T¹-NR^(C)— where R^(B) and R^(C) are each independently H or—C₁-C₈ alkyl,—C(O)hetC(O)— wherein het is a monocyclic heteroaryl of 5 to 12 members,containing one or two heteroatoms independently selected from O, N andS, wherein het is optionally substituted with 1 to 8 substituents eachindependently selected from the group consisting of —C₁-C₈ alkyl, —NH₂,and —NH₂, and said optional substituents on het are optionallysubstituted with —C₁-C₈ alkyl, and—C(A¹)X¹-T²-X¹C(B¹)—, where T² is:

wherein each X¹ is a bond, wherein A1 and B1 are each independently ═O,wherein R¹, R², R³, and R⁴ are each independently H or R¹ and R² form aring system, or R³ and R⁴ form a ring system, or both R¹ and R², and R³and R⁴, each independently form ring systems, or R¹ and R³ form a ringsystem, or R² and R⁴ form a ring system, or both R¹ and R³, and R² andR⁴, each independently form ring systems, where said ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₀ carbocyclycl,and wherein D is a bond or is selected from the group consisting of —S—,—C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₁₀heterocyclo and —C₃-C₈ carbocyclo, where said —C₁-C₈ alkylene-, —C₆-C₁₄arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₁₀ heterocyclo and —C₃-C₈carbocyclo are optionally substituted with —NH₂, —N(R)C(O)H or—N(R)C(O)OH.

Additional aspects of the invention include compounds such as thosementioned herein where

each R is independently selected from the group consisting of H,deuterium, —C₁-C₂₀ alkyl and —NH₂;

each V¹ is independently O or N(R) for each ring system in which V¹appears;

each V² is independently O or N(R) for each ring system in which V²appears;

W¹ and W² are each independently H, —C₁-C₅ alkyl, —C(O)OR, or —C(O)NR₂for each ring system in which W¹ and W² appear;

each X is independently halo, for each ring system in which X appears;

each Y is independently selected from the group consisting of H,—C(O)R^(A), —C(O)N(R^(A))₂, a carbohydrate such as glycosyl, —NO₂,—PO(OR^(A))₂, an amino acid, and a peptide (in particular a peptide thatis cleaved by proteases such as cathepsins and matrixmetalloproteinases) for each ring system in which Y appears, whereineach R^(A) is independently selected from the group consisting of H,—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₃-C₈ carbocyclyl and —C₁-C₂₀alkylN(R)₂, wherein said —C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₃-C₈carbocyclyl and —C₁-C₂₀ alkylN(R)₂ are optionally substituted with 1 to3 substitutents independently selected from R;L¹ and L² are each independently selected from a direct bond andcarbonyl; andT is —C(A¹)X¹-T²-X^(C)(B¹)—, where T² is:

wherein each X¹ is a bond, wherein A1 and B1 are each independently ═O,wherein R¹, R², R³, and R⁴ are each independently H or R¹ and R² form aring system, or R³ and R⁴ form a ring system, or both R¹ and R², and R³and R⁴, each independently form ring systems, or R¹ and R³ form a ringsystem, or R² and R⁴ form a ring system, or both R¹ and R³, and R² andR⁴, each independently form ring systems, where said ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₈ carbocyclycl,and wherein D is a bond or is selected from the group consisting of —S—,—C₁-C₈ alkylene-, —C₆-C₁₄ arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₁₀heterocyclo and —C₃-C₈ carbocyclo, where said —C₁-C₈ alkylene-, —C₆-C₁₄arylene-, —C₆-C₁₄ heteroarylene-, —C₁-C₁₀ heterocyclo and —C₃-C₈carbocyclo are optionally substituted with —NH₂, —N(R)C(O)H or—N(R)C(O)OH.

Additional aspects of the invention include compounds such as thosementioned herein where

L^(A) is selected from the group consisting of -halo, —N(R)₂, —CON(R)₂,—S-aryl optionally substituted with —NO₂ or —CON(R)₂, —S-heteroaryloptionally substituted with —NO₂, alkyl-SO₂-heteroaryl,arylSO₂-heteroaryl-, and

L^(B) is L^(B1)-L^(B2)-L^(B3) wherein L^(B1) is absent or is one or morecomponents selected from the group consisting of —C(O)—, —C(S)—,—C(O)NR—, —C(O)C₁-C₆alkyl-, —C(O)NRC₁-C₆alkyl-,—C₁-C₆alkyl(OCH₂CH₂)₁₋₅—, —C(O)C₁-C₆alkylNRC(O)—,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₅—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₅—C(O)—,—C₁-C₆alkyl-S—S—C₁-C₆alkylNRC(O)CH₂—,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NR^(C)(O)CH₂—,—C(O)C₁-C₆alkyl-NR^(C)(O)C₁₋₆alkyl-,—C(O)—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NRC(O)—,—C(O)C₁-C₆alkyl-phenyl(NR—C(O)C₁-C₆alkyl)₁₋₄-,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₅—NR^(C)(O)C₁-C₆alkyl-, —C₁-C₆alkyl-, —S—,—C(O)—CH(NR—C(O)C₁-C₆alkyl)-C₁-C₆alkyl- and (—CH₂—CH₂—O—)₁₋₂₀, whereinL^(B2) is AA₀₋₁₂, wherein AA is a natural amino acid, a non-naturalamino acid or —(CR¹⁵)_(o)—S—S—(CR¹⁵)_(p) where o and p are eachindependently an integer from 1 to 20, and L^(B3) is —PABA-, —PABC—,—C(O)(CH₂)_(n)C(O)— or absent; and L^(C) is absent.

Additional aspects of the invention include antibody drug conjugatessuch as those mentioned herein where L^(A) is selected from: a bond toAB, —NR-(bond to AB), -heteroaryl-(bond to AB),

L^(B) is L^(B1)-L^(B2)-L^(B3) wherein L^(B1) is absent or is one or morecomponents selected from the group consisting of —C(O)—, —C(S)—,—C(O)NR—, —C(O)C₁-C₆alkyl-, —C(O)NRC₁-C₆alkyl-,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C(O)C₁-C₆alkylNRC(O)—,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆—C(O)—,—C₁-C₆alkyl-S—S—C₁-C₆alkylNRC(O)CH₂—,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NR^(C)(O)CH₂—,—C(O)C₁-C₆alkyl-NR^(C)(O)C₁₋₆alkyl-,—C(O)—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NR^(C)(O)—,—C(O)C₁-C₆alkyl-phenyl(NR—C(O)C₁-C₆alkyl)₁₋₄-,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—NR^(C)(O)C₁-C₆alkyl-, —C₁-C₆alkyl-, —S—,—C(O)—CH(NR—C(O)C₁-C₆alkyl)-C₁-C₆alkyl- and (—CH₂—CH₂—O—)₁₋₂₀, whereinL^(B2) is AA₀₋₁₂, wherein AA is a natural amino acid, a non-naturalamino acid or —(CR¹⁵)_(o)—S—S—(CR¹⁵)_(p) where o and p are eachindependently an integer from 1 to 20, and L^(B3) is —PABA-, —PABC—,—C(O)(CH₂)_(n)C(O)— or absent.

Additional aspects of the invention include compounds such as thosementioned herein where R^(F) is selected from:

wherein q is 1-10, and each b is independently CR^(D), N, NR^(D), O orS.

Additional aspects of the invention include compounds such as thosementioned herein where one or more W is C₁-C₃ alkyl.

Additional aspects of the invention include compounds such as thosementioned herein where X is chloro.

Additional aspects of the invention include compounds such as thosementioned herein where one Y is H or —C(O)C₁-C₁₀alkyl.

Additional aspects of the invention include compounds such as thosementioned herein where one or more Z is H.

Additional aspects of the invention include compounds such as thosementioned herein where T is selected from an amide, oramino-tether-amino of the formula —NH—C(O)—NH— or —NH—C(O)-het-C(O)—NH—.

Additional aspects of the invention include compounds such as thosementioned herein where the amide is —C(O)NH— or —NHC(O)—.

Additional aspects of the invention include compounds such as thosementioned herein where het is a heteroaryl selected frompyrrol-2-,5-diyl-, fur-2,5-diyl-, indol-2,5-diyl, benzofuran-2,5-diyl,and 3, 6-dihydrobenzo[1, 2-b:4, 3-b]dipyrrol-2,7-diyl.

Additional aspects of the invention include compounds such as thosementioned herein where L¹ and L² are selected from carbonyl,2-carbonylindole-5-yl, 2-carbonyl-6-hydroxy-7-methoxyindol-5-yl,2-carbonyl-1,2,3,6-tetrahydrobenzo[1,2-b:4,3-b]dipyrrol-7-yl,2-carbonyl-4-hydroxy-5-methoxy-1,2,3,6-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-7-yl,and2-carbonyl-4-hydroxy-5-methoxy-1,2,3,6-tetrahydrobenzo[1,2-b:4,3-b′]dipyrrol-7-yl.

Additional aspects of the invention are those compounds recited hereinwhere one or more of the following apply: W is methyl; X is a halogen; Yis hydrogen or —COR where R is C₁-C₁₀alkyl; and Z is hydrogen.

The invention also includes compound as described herein where T isselected from an amide (i.e., —C(O)NH— or —NHC(O)—); or anamino-tether-amino of the formula —NH-T′-NH where T′ is carbonyl or—C—(O)-het-C(O)—. Where T is an amino-tether-amino of the formulaNH-T′-NH, T′ may be carbonyl (i.e., —C—(O)—) or —C(O)-het-C(O)— wherehet is a heteroaryl selected from pyrrol-2-, 5-diyl-; fur-2, 5-diyl-;indol-2, 5-diyl; benzofuran-2, 5-diyl; or 3, 6-dihydrobenzo[1, 2-b:4,3-b]dipyrrol-2, 7-diyl.

Also included in embodiments of the invention are those compounds asdescribed herein where L¹ and L² are selected from2-carbonylindole-5-yl; 2-carbonyl-6-hydroxy-7-methoxyindol-5-yl;2-carbonyl-1, 2, 3, 6-tetrahydrobenzo[1, 2-b:4, 3-b]dipyrrol-7-yl;2-carbonyl-4-hydroxy-5-methoxy-1, 2, 3, 6-tetrahydrobenzo[1, 2-b:4,3-b′]dipyrrol-7-yl; and 2-carbonyl-4-hydroxy-5-methoxy-1, 2, 3,6-tetrahydrobenzo[1, 2-b:4, 3-b′ ]dipyrrol-7-yl.

Another aspect of the invention includes compounds as described hereinwhere L^(A) is

The invention includes, as well, linker-payloads or anantibody-drug-conjugates comprising a radical of the payload compoundsdescribed herein.

Importantly, the invention includes pharmaceutical compositions of thecompounds, and any pharmaceutically acceptable salts or solvatesthereof, described herein, where the pharmaceutical composition includesa pharmaceutically acceptable excipient.

The invention further relates to methods of treating cancer comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a one or more of compound described herein, or apharmaceutical composition or compositions comprising one or more ofthese compounds.

Some compounds, including payloads, linker-payloads and ADCs depictedherein, are shown in a specific stereoisomeric form. The invention,however, is meant to include all stereoisomeric forms of thesecompounds. For instance, a compound with two stereoisomeric centers maybe depicted as the R, S form of the compound, but the invention conveysall stereoisomeric forms, e.g., R,R; R,S; S,R and S,S.

DETAILED DESCRIPTION

The present invention is directed to cytotoxic bifunctional compounds,to antibody drug conjugates (ADCs) comprising said cytotoxicbifunctional compounds, and to methods for using the same to treatcancer and other pathological conditions. The invention also relates tomethods of using such compounds and/or conjugates in vitro, in situ, andin vivo for the detection, diagnosis or treatment of mammalian cells, orassociated pathological conditions.

Definitions and Abbreviations

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings. When trade names are usedherein, the trade name includes the product formulation, the genericdrug, and the active pharmaceutical ingredient(s) of the trade nameproduct, unless otherwise indicated by context.

The term “antibody” (or “Ab”) herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,monospecific antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments that exhibit the desired biologicalactivity. An intact antibody has primarily two regions: a variableregion and a constant region. The variable region binds to and interactswith a target antigen. The variable region includes a complementarydetermining region (CDR) that recognizes and binds to a specific bindingsite on a particular antigen. The constant region may be recognized byand interact with the immune system (see, e.g., Janeway et al., 2001,Immuno. Biology, 5th Ed., Garland Publishing, New York). An antibody canbe of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The antibody can bederived from any suitable species. In some embodiments, the antibody isof human or murine origin. An antibody can be, for example, human,humanized or chimeric.

The terms “specifically binds” and “specific binding” refer to antibodybinding to a predetermined antigen. Typically, the antibody binds withan affinity of at least about 1×10⁷ M⁻¹, and binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method.

The term “monoclonal antibodies” specifically includes “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical to or homologous with the corresponding sequence of antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical toor homologous with the corresponding sequences of antibodies derivedfrom another species or belonging to another antibody class or subclass,as well as fragments of such antibodies, so long as they exhibit thedesired biological activity.

An “intact antibody” is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H1), C_(H2), C_(H3) and C_(H4), as appropriate forthe antibody class. The constant domains may be native sequence constantdomains (e.g., human native sequence constant domains) or amino acidsequence variants thereof.

An intact antibody may have one or more “effector functions”, whichrefers to those biological activities attributable to the Fc region(e.g., a native sequence Fc region or amino acid sequence variant Fcregion) of an antibody. Examples of antibody effector functions includecomplement dependent cytotoxicity, antibody-dependent cell-mediatedcytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis.

An “antibody fragment” comprises a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments, diabodies, triabodies, tetrabodies, linear antibodies,single-chain antibody molecules, scFv, scFv-Fc, multispecific antibodyfragments formed from antibody fragment(s), a fragment(s) produced by aFab expression library, or an epitope-binding fragments of any of theabove which immuno specifically bind to a target antigen (e.g., a cancercell antigen, a viral antigen or a microbial antigen).

The term “variable” in the context of an antibody refers to certainportions of the variable domains of the antibody that differ extensivelyin sequence and are used in the binding and specificity of eachparticular antibody for its particular antigen. This variability isconcentrated in three segments called “hypervariable regions” in thelight chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FRs). The variable domains of native heavy and light chains eachcomprise four FRs connected by three hypervariable regions.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (L3) in the heavy chain variabledomain; Kabat et al. (Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (e.g.,residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (142) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, 1987, J. Mol. Biol.196:901-917). FR residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

A “single-chain Fv” or “scFv” antibody fragment comprises the V.sub.Hand V.sub.L domains of an antibody, wherein these domains are present ina single polypeptide chain. Typically, the Fv polypeptide furthercomprises a polypeptide linker between the V.sub.H and V.sub.L domainswhich enables the scFv to form the desired structure for antigenbinding. For a review of scFv, see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabody” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 0 404 097; WO 93/11161; and Hollinger et al., 1993, Proc.Natl. Acad. Sci. USA 90:6444-6448.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., 1986, Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta,1992, Curr. Op. Struct. Biol. 2:593-596.

used herein, “isolated” means separated from other components of (a) anatural source, such as a plant or animal cell or cell culture, or (b) asynthetic organic chemical reaction mixture. As used herein, “purified”means that when isolated, the isolate contains at least 95%, and inanother aspect at least 98%, of a compound (e.g., a conjugate) by weightof the isolate.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is a tumor cell, e.g., a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic or bladdercell. Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering; and nuclear/chromatin condensationalong with DNA fragmentation can be evaluated by any increase inhypodiploid cells.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may inhibit the growth of and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic. For cancertherapy, efficacy can, for example, be measured by assessing the time todisease progression (TTP) and/or determining the response rate (RR).

The term “substantial amount” refers to a majority, i.e. greater than50% of a population, of a mixture or a sample.

The term “intracellular metabolite” refers to a compound resulting froma metabolic process or reaction inside a cell on an antibody-drugconjugate (ADC). The metabolic process or reaction may be an enzymaticprocess such as proteolytic cleavage of a peptide linker of the ADC.Intracellular metabolites include, but are not limited to, antibodiesand free drug which have undergone intracellular cleavage after entry,diffusion, uptake or transport into a cell.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on an ADC or the like,whereby the covalent attachment, e.g., the linker, between the drugmoiety and the antibody is broken, resulting in the free drug, or othermetabolite of the conjugate dissociated from the antibody inside thecell. The cleaved moieties of the ADC are thus intracellularmetabolites.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of a drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The term “cytotoxic activity” refers to a cell-killing, a cytostatic oran anti-proliferative effect of a ADC or an intracellular metabolite ofsaid ADC. Cytotoxic activity may be expressed as the IC₅₀ value, whichis the concentration (molar or mass) per unit volume at which half thecells survive.

A “disorder” is any condition that would benefit from treatment with adrug or antibody-drug conjugate. This includes chronic and acutedisorders or diseases including those pathological conditions whichpredispose a mammal to the disorder in question. Non-limiting examplesof disorders to be treated herein include benign and malignant cancers;leukemia and lymphoid malignancies, neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; and inflammatory, angiogenic and immunologicdisorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition or disorder in mammals that is typicallycharacterized by unregulated cell growth. A “tumor” comprises one ormore cancerous cells.

Examples of a “patient” include, but are not limited to, a human, rat,mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird andfowl. In an exemplary embodiment, the patient is a human.

The terms “treat” or “treatment,” unless otherwise indicated by context,refer to therapeutic treatment and prophylactic measures to preventrelapse, wherein the object is to inhibit or slow down (lessen) anundesired physiological change or disorder, such as the development orspread of cancer. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadyhaving the condition or disorder as well as those prone to have thecondition or disorder.

In the context of cancer, the term “treating” includes any or all ofinhibiting growth of tumor cells, cancer cells, or of a tumor;inhibiting replication of tumor cells or cancer cells, lessening ofoverall tumor burden or decreasing the number of cancerous cells, andameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includesany or all of inhibiting replication of cells associated with anautoimmune disease state including, but not limited to, cells thatproduce an autoimmune antibody, lessening the autoimmune-antibody burdenand ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includesany or all of: inhibiting the growth, multiplication or replication ofthe pathogen that causes the infectious disease and ameliorating one ormore symptoms of an infectious disease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indication(s), usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

As used herein, the terms “cell,” “cell line,” and “cell culture” areused interchangeably and all such designations include progeny. Thewords “transformants” and “transformed cells” include the primarysubject cell and cultures or progeny derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

As used herein, CBI refers to1,2,9,9a-tetrahydro-4H-benzo[e]cyclopropa[c]indol-4-one, or asubstituted or derivatized form thereof. CBI can also refer to the secoform of CBI, or seco-CBI, which is also know as1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol, or a substituted orderivatized form (or forms) thereof.

As used herein, CPI refers to1,2,8,8a-tetrahydrocyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one or asubstituted or derivatized form thereof. CPI can also refer to the secoform of CPI, or seco-CPI, which is also know as8-(chloromethyl)-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-ol, ora substituted or derivatized form (or forms) thereof.

Unless otherwise indicated, the term “alkyl” by itself or as part ofanother term refers to a straight chain or branched, saturatedhydrocarbon having the indicated number of carbon atoms (e.g., “C₁-C₈”alkyl refer to an alkyl group having from 1 to 8 carbon atoms). Alkylgroups typically comprise from 1 to 20 carbon atoms, preferably from 1to 8 carbon atoms, and more preferably from 1 to 4 carbon atoms. Whenthe number of carbon atoms is not indicated, the alkyl group has from 1to 8 carbon atoms. Representative straight chain C₁-C₈ alkyls include,but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl,n-hexyl, n-heptyl and n-octyl; while branched C₁-C₈ alkyls include, butare not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl,-isopentyl, and -2-methylbutyl; unsaturated C₂-C₈ alkyls include, butare not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl,1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl and 3-methyl-1-butynyl.Reference to “alkyl” herein refers to unsubstituted and substitutedmoieties as described above.

Unless otherwise indicated, “alkylene,” by itself of as part of anotherterm, refers to a saturated, branched or straight chain or cyclichydrocarbon radical of the stated number of carbon atoms, typically 1-18carbon atoms, and having two monovalent radical centers derived by theremoval of two hydrogen atoms from the same or two different carbonatoms of a parent alkane.) Alkylene groups typically comprise from 1 to18 carbon atoms, preferably from 1 to 10 carbon atoms, more preferablyfrom 1 to 8 carbon atoms, and most preferably from 1 to 4 carbon atoms.Typical alkylene radicals include, but are not limited to: methylene(—CH₂—), 1,2-ethylene —CH₂CH₂—), 1,3-propylene (—CH₂CH₂CH₂—),1,4-butylene (—CH₂CH₂CH₂CH₂—), and the like. A “C₁-C₁₀” straight chainalkylene is a straight chain, saturated hydrocarbon group of the formula—(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene include methylene, ethylene,propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonyleneand decalene. Reference to “alkylene” herein refers to unsubstituted andsubstituted moieties as described above.

Unless otherwise indicated, the term “heteroalkyl,” by itself or incombination with another term, means, unless otherwise stated, a stablestraight or branched chain hydrocarbon, or combinations thereof, fullysaturated or containing from 1 to 3 degrees of unsaturation, consistingof the stated number of carbon atoms and from one to three heteroatomsselected from the group consisting of O, N, Si and S, and wherein thenitrogen and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N and Smay be placed at any interior position of the heteroalkyl group. Theheteroatom Si may be placed at any position of the heteroalkyl group,including the position at which the alkyl group is attached to theremainder of the molecule. Up to two heteroatoms may be consecutive.Heteroalkyl groups typically comprise from 1 to 15 carbon atoms,preferably from 1 to 12 carbon atoms, more preferably from 1 to 8 carbonatoms, and most preferably from 1 to 4 carbon atoms. Reference to“heteroalkyl” herein refers to unsubstituted and substituted moieties asdescribed above.

Unless otherwise indicated, the term “heteroalkylene” by itself or aspart of another substituent means a divalent group derived fromheteroalkyl (as discussed above). For heteroalkylene groups, heteroatomscan also occupy either or both of the chain termini. Reference to“heteroalkylene” herein refers to unsubstituted and substituted moietiesas described above.

The term H or hydrogen herein typically refers to a hydrogen atomcomprising a single proton and no neutron, but also includes thehydrogen isotope known as deuterium which comprises a single proton anda single neutron.

Unless otherwise indicated, “aryl,” by itself or an part of anotherterm, means a substituted or unsubstituted monovalent carbocyclicaromatic hydrocarbon radical of 5-20, preferably 5-14 or 6-14, carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Typical aryl groups include, butare not limited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like. A substitutedcarbocyclic aromatic group (e.g., an aryl group) can be substituted withone or more, preferably 1 to 5, of the following groups: C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), —C(O)R⁹, —OC(O)R⁹, —C(O)OR⁹, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂,—NH(R⁹), —N(R⁹)₂ and —CN; wherein each R⁹ is independently selected from—H, C₁-C₈ alkyl and unsubstituted aryl. In some embodiments, asubstituted carbocyclic aromatic group can further include one or moreof: —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R⁹ and —SR⁹. “Arylene” is thecorresponding divalent moiety.

“Substituted alkyl” (or “substituted alkylene”, “substitutedheteroalkyl”, or “substituted heteroalkylene”) means an the relevantalkyl alkyl-containing group or radical as discussed above in which oneor more hydrogen atoms are each independently replaced with asubstituent. Typical substituents include, but are not limited to, —X,—R¹⁰, —O—, —OR¹⁰, —SR¹⁰, —S—, —NR¹⁰ ₂, —NR¹⁰ ₃, ═N R¹⁰, —CX₃, —CN, —OCN,—SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, —N R¹⁰C(═O)R¹⁰R¹⁰, —C(═O)NR¹⁰₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R¹⁰, —OS(═O)₂OR¹⁰, —S(═O)₂NR¹⁰, —S(═O)R¹⁰,—OP(═O)(OR¹⁰)₂, —P(═O)(OR¹⁰)₂, —PO₃ ²⁻, PO₃H₂, —AsO₂H₂, —C(═O)R¹⁰,—C(═O)X, —C(═S) R¹⁰, —CO₂R¹⁰, —CO₂ ⁻, —C(═S)OR¹⁰, —C(═O)SR¹⁰,—C(═S)SR¹⁰, —C(═O)NR¹⁰ ₂, —C(═S)NR¹⁰ ₂, or —C(═N R¹⁰)N R¹⁰ ₂, where eachX is independently a halogen: —F, —C, —Br, or —I; and each R¹⁰ isindependently —H, C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl, C₆-C₂₀ aryl, C₁-C₁₀heterocyclyl, a protecting group or a prodrug moiety. Aryl, alkylene andheteroalkylene groups as described above may also be similarlysubstituted.

Unless otherwise indicated, “aralkyl” by itself or part of another term,means an alkyl group, as defined above, substituted with an aryl group,as defined above.

Unless otherwise indicated, “C₃-C₁₀ heterocyclyl” by itself or as partof another term, refers to a monovalent substituted or unsubstitutedaromatic or non-aromatic monocyclic, bicyclic or tricyclic ring systemhaving from 2 to 10, 2 to 14, or 2-20 carbon atoms, preferably 3 to 8,carbon atoms (also referred to as ring members) and one to fourheteroatom ring members independently selected from N, O, P or S, andderived by removal of one hydrogen atom from a ring atom of a parentring system. One or more N, C or S atoms in the heterocyclyl can beoxidized. The ring that includes the heteroatom can be aromatic ornonaromatic. Aromatic heterocycles are sometimes referred to herein asheteroaryls. Unless otherwise noted, the heterocyclyl is attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure. Representative examples of a C₂-C₁₀ heterocyclyl include, butare not limited to, tetrahyrofuranyl, oxetanyl, pyranyl, pyrrolidinyl,piperidinyl, piperazinyl, benzofuranyl, benzothiophene, benzothiazolyl,indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiopene), furanyl,thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl includingmoieties such as 1,2,3,4-tetrshyhro-quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, tetrazolyl,epoxide, oxetane and BODIPY (substituted or unsubstituted). A C₂-C₁₀heterocyclyl can be substituted with up to seven groups including, butnot limited to, C₁-C₈ alkyl, C₁-C₈ heteroalkyl, —OR¹¹, aryl, —C(O)R¹¹,—OC(O)R¹¹, —C(O)OR¹¹, —C(O)NH₂, —C(O)NHR¹¹, —C(O)N(R¹¹)₂, —NHC(O)R¹¹,—S(═O)₂R¹¹, —S(O)R¹¹, halogen, —N₃, —NH₂, —NH(R¹¹), —N(R¹¹)₂ and —CN;wherein each R¹¹ is independently selected from —H, C₁-C₈ alkyl, C₁-C₈heteroalkyl and aryl. In some embodiments, a substituted heterocyclylcan also include one or more of: —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R¹¹ and—SR¹¹. Heterocyclo or C₂-C₁₀ heterocyclo is the corresponding divalentmoiety. Divalent aromatic heterocycles are sometimes referred to hereinas heteroarylene or C₂-C₁₀ heteroarylene.

As noted above, aromatic heterocycles are sometimes referred to hereinas heteroaryls, and preferably contain 5-14, 6-14, or 6-20 carbon atomsin addition to heteroatoms. Heteroaryls may be monocyclic, bicyclic, ortricyclic ring systems. Representative heteroaryls include but are notlimited to triazolyl, tetrazolyl, oxadiazolyl, pyridyl, furyl,benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl,indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl,benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl,quinazolinyl, pyrimidyl, azepinyl, oxepinyl, and quinoxalinyl.Heteroaryls are optionally substituted. Typical substituents include,but are not limited to, —X, —R^(h), —O—, —OR^(h), —SR^(h), —S⁻, —NR^(h)₂, —NR^(h) ₃, ═NR^(h), —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂,═N₂, —N₃, —NR^(h)C(═O)R^(h), —C(═O)NR^(h) ₂, —SO₃ ⁻, —SO₃H, —S(═O)₂Rh,—OS(═O)₂OR^(h), —S(═O)₂NR^(h), —S(═O)R^(h), —OP(═O)(OR^(h))₂,—P(═O)(OR^(h))₂, —PO₃ ²⁻, PO₃H₂, —AsO₂H₂, —C(═O)R^(h), —C(═O)X,—C(═S)R^(h), —CO₂Rh, —C₀₂—, —C(═S)OR^(h), —C(═O)SR^(h), —C(═S)SR^(h),—C(═O)NR^(h) ₂, —C(═S)NR^(h) ₂, —C(═NR)NR^(h) ₂, C₁-C₂₀ heteroalkyl,C₆-C₂₀ aryl, C₃-C₈ heterocyclyl, a protecting group or a prodrug moiety,where each X is independently a halogen: —F, —Cl, —Br, or —I; and eachR^(h) is independently —H or C₁-C₈ alkyl. Divalent aromatic heterocyclesare sometimes referred to herein as heteroarylenes or C₁-C₁₀heteroarylenes.

Unless otherwise indicated, “heteroaralkyl” by itself or part of anotherterm, means an alkyl group, as defined above, substituted with anaromatic heterocyclyl group, as defined above. Heteroaralklo is thecorresponding divalent moiety.

Unless otherwise indicated, “C₃-C₈ carbocyclyl” by itself or as part ofanother term, is a 3-, 4-, 5-, 6-, 7- or 8-membered monovalent,substituted or unsubstituted, saturated or unsaturated non-aromaticmonocyclic or bicyclic carbocyclic ring derived by the removal of onehydrogen atom from a ring atom of a parent ring system. RepresentativeC₃-C₈ carbocyclyl include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl,1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl,cyclooctadienyl, bicyclo(1.1.1.)pentane, and bicyclo(2.2.2.)octane. AC₃-C₈ carbocyclyl group can be unsubstituted or substituted with up toseven groups including, but not limited to, C₁-C₈ alkyl, C₁-C₈heteroalkyl, —OR¹¹, aryl, —C(O)R¹¹, —OC(O)R¹¹, —C(O)OR¹¹, —C(O)NH₂,—C(O)NHR¹¹, —C(O)N(R¹¹)₂, —NHC(O)R¹¹, —S(═O)₂R¹¹, —S(═O)R¹¹, —OH,-halogen, —N₃, —NH₂, —NH(R¹¹), —N(R¹¹)₂ and —CN; where each R¹¹ isindependently selected from —H, C₁-C₈ alkyl, C₁-C₈ heteroalkyl and aryl.“C₃-C₈ carbocyclo” is the corresponding divalent moiety.

As used herein, an azido substituent refers to —N═N═N; a cyanatesubstituent refers to —OCN; a thiocyanate substituent refers to —S—CN;an isocyanate substituent refers to —N═C═O; and a thioisocyanatesubstituent refers to —S—N═C═O.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g., melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Glycosyl” refers to the structure:

or substituted forms of same, for instance including the referencesstructure substituted to form structures such as:

and many others.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms, McGraw-HillBook Company, New York (1984); and Eliel and Wilen, Stereochemistry ofOrganic Compounds, John Wiley & Sons, Inc., New York (1994). Manyorganic compounds exist in optically active forms, i.e., they have theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L, or R and S, are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and l or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or 1 meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesestereoisomers are identical except that they are mirror images of oneanother. A specific stereoisomer may also be referred to as anenantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

As used herein, “—PABA-” or “PABA” refers to the p-aminobenzoic acid andmoieties derived therefrom, for instance the structure:

or variants thereof.

As used herein, “—PABC-” or “PABC” refers to p-aminobenzyloxycarbonyland moieties derived therefrom, for instance the structure:

or variants thereof.

An amino acid “derivative” includes an amino acid having substitutionsor modifications by covalent attachment of a parent amino acid, such as,e.g., by alkylation, glycosylation, acetylation, phosphorylation, andthe like. Further included within the definition of “derivative” is, forexample, one or more analogs of an amino acid with substituted linkages,as well as other modifications known in the art.

A “natural amino acid” refers to arginine, glutamine, phenylalanine,tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine,proline, glutamic acid, aspartic acid, threonine, cysteine, methionine,leucine, asparagine, isoleucine, and valine, unless otherwise indicatedby context.

“Protecting group” refers to a moiety that when attached to a reactivegroup in a molecule masks, reduces or prevents that reactivity. Examplesof protecting groups can be found in T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons,New York, 1999, and Harrison and Harrison et al., Compendium ofSynthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996),which are incorporated herein by reference in their entirety.Representative hydroxy protecting groups include acyl groups, benzyl andtrityl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allylethers. Representative amino protecting groups include, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl(Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES),trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC),and the like.

Examples of a “hydroxyl protecting group” include, but are not limitedto, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranylether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether,triethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silylether, triphenylmethyl silyl ether, acetate ester, substituted acetateesters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Such leaving groups are well known in the art,and examples include, but are not limited to, a halide (e.g., chloride,bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),trifluoromethylsulfonyl(triflate), and trifluoromethylsulfonate.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound.The compound typically contains at least one amino group, andaccordingly acid addition salts can be formed with this amino group.Exemplary salts include, but are not limited to, sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, malate, gentisinate, fumarate, gluconate,glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and a compound or conjugateof the invention. Examples of solvents that form pharmaceuticallyacceptable solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The terms “loading” or “drug loading” or “payload loading” represent orrefer to the average number of payloads (“payload” and “payloads” areused interchangeable herein with “drug” and “drugs”) per antibody in anADC molecule. Drug loading may range from I to 20 drugs per antibody.This is sometimes referred to as the DAR, or drug to antibody ratio.Compositions of the ADCs described herein typically have DAR's of from1-20, and in certain embodiments from 1-8, from 2-8, from 2-6, from 2-5and from 2-4. Typical DAR values are 2, 4, 6 and 8. The average numberof drugs per antibody, or DAR value, may be characterized byconventional means such as UV/visible spectroscopy, mass spectrometry,ELISA assay, and HPLC. The quantitative DAR value may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADCs having a particular DAR value maybe achieved by means such as reverse phase HPLC or electrophoresis. DARmay be limited by the number of attachment sites on the antibody. Forexample, where the attachment is a cysteine thiol, an antibody may haveonly one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a Linker unitmay be attached. In some embodiments, the cysteine thiol is a thiolgroup of a cysteine residue that forms an interchain disulfide bond. Insome embodiments, the cysteine thiol is a thiol group of a cysteineresidue that does not form an interchain disulfide bond. Typically,fewer than the theoretical maximum of drug moieties are conjugated to anantibody during a conjugation reaction. An antibody may contain, forexample, many lysine residues that do not react with a linker or linkerintermediate. Only the most reactive lysine groups may react with areactive linker reagent.

Generally, antibodies do not contain many, if any, free and reactivecysteine thiol groups which may be linked to a drug via a linker. Mostcysteine thiol residues in the antibodies exist as disulfide bridges andmust be reduced with a reducing agent such as dithiothreitol (DTT). Theantibody may be subjected to denaturing conditions to reveal reactivenucleophilic groups such as lysine or cysteine. The loading(drug/antibody ratio) of an ADC may be controlled in several differentmanners, including: (i) limiting the molar excess of drug-linkerrelative to the antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification. Where more than one nucleophilic groupreacts with a drug-linker then the resulting product is a mixture ofADCs with a distribution of one or more drugs moieties per antibody. Theaverage number of drugs per antibody may be calculated from the mixtureby, for example, dual ELISA antibody assay, specific for antibody andspecific for the drug. Individual ADCs may be identified in the mixtureby mass spectroscopy, and separated by HPLC, e. g, hydrophobicinteraction chromatography.

Below is a list of abbreviations and definitions that may not otherwisebe defined or described in this application: DMSO (refers to dimethylsulfoxide), HRMS (refers to high resolution mass spectrometry), DAD(refers to diode array detection), TFA (refers to 2,2,2-trifluoroaceticacid or trifluoroacetic acid), TFF (refers to tangential flowfiltration), EtOH (refers to ethanol), MW (refers to molecular weight),HPLC (refers to high performance liquid chromatography), prep HPLC(refers to preparative high performance liquid chromatography), etc.(refers to and so forth), trityl (refers1,1′,1″-ethane-1,1,1-triyltribenzene), THF (refers to tetrahydrofuran),NHS (refers to 1-Hydroxy-2,5-pyrrolidinedione), Cbz (refers tocarboxybenzyl), eq. (refers to equivalent), n-BuLi (refers ton-butyllithium), OAc (refers to acetate), MeOH (refers to methanol),i-Pr (refers to isopropyl or propan-2-yl), NMM (refers to4-methylmorpholine), and “-” (in a table refers to no data available atthis time).

Divalent moieties and substituents used herein are meant to refer tosaid moieties or substituents bound or linked in either direction orboth directions. For instance, the moiety —C(O)NR— (in the definition ofL^(B1), and elsewhere) is meant to convey —C(O)NR— as well as—NR^(C)(O)—, the moiety —C(O)C₁-C₆alkyl- is meant to convey—C(O)C₁-C₆alkyl- as well as —C₁-C₆alkylC(O)—, and so on. More generally,a description of a non-symmetrical divalent moiety linked on its “left”and “right” sides is meant to convey both the moiety as presented (leftside of the moiety linked on left side as written, right side of themoiety linked on the right side as written) and the reverse of themoiety as presented (left side of the moiety linked on right side aswritten, right side of the moiety linked on the left side as written).

The terms “bond” and “absent” are both used herein to describe avariable which does not include an atom or atoms. Thus, where a divalentvariable that is “absent” is understood to mean that the adjacentmoieties are bound to one another. For example, if L^(B2) is absent itis understood that L^(B1) may be bound to L^(B3); or if L^(B1) andL^(B2) are both absent it is understood that L^(A) may be bound toL^(B3). Similarly, if a divalent variable is defined as being a “bond”this is understood to mean that there are no atoms present and theadjacent moieties are bound to one another. Thus, for instance, wherevariable “D” is defined as being a bond it is appreciated that thecarbons adjacent D (in the structure defining T²) are bound to oneanother. An absent monovalent variable is understood to be a hydrogen oran electron pair capable of further covalent bonding.

The Antibody Unit (A, Ab or AB)

As noted above, the term “antibody” (or “A”, “Ab” or “AB”) herein isused in the broadest sense and specifically covers intact monoclonalantibodies, polyclonal antibodies, monospecific antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments that exhibit the desired biological activity. In addition,while certain aspects of the invention described herein refer toantibody drug conjugates, it is further envisioned that the antibodyportion of the conjugate might be replaced with anything thatspecifically binds or reactively associates or complexes with areceptor, antigen or other receptive moiety associated with a giventarget-cell population. For example, instead of containing an antibody aconjugates of the invention could contain a targeting molecule thatbinds to, complexes with, or reacts with a receptor, antigen or otherreceptive moiety of a cell population sought to be therapeutically orotherwise biologically modified. Example of such molecules includesmaller molecular weight proteins, polypeptide or peptides, lectins,glycoproteins, non-peptides, vitamins, nutrient-transport molecules(such as, but not limited to, transferrin), or any other cell bindingmolecule or substances. In certain aspects, the antibody or other suchtargeting molecule acts to deliver a drug to the particular target cellpopulation with which the antibody or other targeting moleculeinteracts.

In another aspect, the present invention relates to an antibody drugconjugate compound of Formulae IIIA or IIIB wherein the antibody AB isselected from: trastuzumab, trastuzumab mutants (for instance thetrastuzumab mutants disclosed herein or in international patentapplication PCT/IB2012/056234), oregovomab, edrecolomab, cetuximab, ahumanized monoclonal antibody to the vitronectin receptor (α_(v)β₃),alemtuzumab, anti-HLA-DR antibodies including a humanized anti-HLA-DRantibody for the treatment of non-Hodgkin's lymphoma, 1311 Lym-1,anti-HLA-Dr10 antibodies including a murine anti-HLA-Dr10 antibody forthe treatment of non-Hodgkin's lymphoma, anti-cd33 antibodies, anti-cd22antibodies including a humanized anti-CD22 mAb for the treatment ofHodgkin's Disease or non-Hodgkin's lymphoma, labetuzumab, bevacizumab,ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab,ipilimumab, and gemtuzumab.

Heteroatoms that may be present on an antibody unit include sulfur (inone embodiment, from a sulfhydryl group of an antibody), oxygen (in oneembodiment, from a carbonyl, carboxyl or hydroxyl group of an antibody)and nitrogen (in one embodiment, from a primary or secondary amino groupof an antibody). These hetero atoms can be present on the antibody inthe antibody's natural state, for example a naturally-occurringantibody, or can be introduced into the antibody via chemicalmodification.

In one embodiment, an antibody unit has a sulfhydryl group and theantibody unit bonds via the sulfhydryl group's sulfur atom.

In another embodiment, the antibody has lysine residues that can reactwith activated esters (such esters include, but are not limited to,N-hydroxysuccinimde, pentafluorophenyl, and p-nitrophenyl esters) andthus form an amide bond consisting of the nitrogen atom of the antibodyunit and a carbonyl.

In yet another aspect, the antibody unit has one or more lysine residuesthat can be chemically modified to introduce one or more sulfhydrylgroups. The reagents that can be used to modify lysines include, but arenot limited to, N-succinimidyl S-acetylthioacetate (SATA) and2-Iminothiolane hydrochloride (Traut's Reagent).

In another embodiment, the antibody unit can have one or morecarbohydrate groups that can be chemically modified to have one or moresulfhydryl groups.

In yet another embodiment, the antibody unit can have one or morecarbohydrate groups that can be oxidized to provide an aldehyde group(see, e.g., Laguzza, et al., 1989, J. Med. Chem. 32(3):548-55). Thecorresponding aldehyde can form a bond with a reactive site such as, forexample, hydrazine and hydroxylamine. Other protocols for themodification of proteins for the attachment or association of drugs aredescribed in Coligan et al., Current Protocols in Protein Science, vol.2, John Wiley & Sons (2002) (incorporated herein by reference).

When the conjugates comprise non-immunoreactive protein, polypeptide, orpeptide units instead of an antibody, useful non-immunoreactive protein,polypeptide, or peptide units include, but are not limited to,transferrin, epidermal growth factors (“EGF”), bombesin, gastrin,gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6,transforming growth factors (“TOP”), such as TGF-α and TGF-β, vacciniagrowth factor (“VGF”), insulin and insulin-like growth factors I and II,somatostatin, lectins and apoprotein from low density lipoprotein.

Useful polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of immunized animals. Useful monoclonalantibodies are homogeneous populations of antibodies to a particularantigenic determinant (e.g., a cancer cell antigen, a viral antigen, amicrobial antigen, a protein, a peptide, a carbohydrate, a chemical,nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to anantigen-of-interest can be prepared by using any technique known in theart which provides for the production of antibody molecules bycontinuous cell lines in culture.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric monoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art(e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312;Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982,Meth. Enzymol. 92:3-16).

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art and are discussed infra.

The antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to target cells (e.g.,cancer cell antigens, viral antigens, or microbial antigens) or otherantibodies that bind to tumor cells or matrix. In this regard,“functionally active” means that the fragment, derivative or analog isable to elicit anti-anti-idiotype antibodies that recognize the sameantigen that the antibody from which the fragment, derivative or analogis derived recognized. Specifically, in an exemplary embodiment theantigenicity of the idiotype of the immunoglobulin molecule can beenhanced by deletion of framework and CDR sequences that are C-terminalto the CDR sequence that specifically recognizes the antigen. Todetermine which CDR sequences bind the antigen, synthetic peptidescontaining the CDR sequences can be used in binding assays with theantigen by any binding assay method known in the art (e.g., the BIA coreassay) (for location of the CDR sequences, see, e.g., Kabat et al.,1991, Sequences of Proteins of Immunological Interest, Fifth Edition,National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J.Immunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, Fab fragments, Fvs, single chainantibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, or anyother molecule with the same specificity as the antibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as for example,those having a variable region derived from a murine monoclonal andhuman immunoglobulin constant regions. (See, e.g., U.S. Pat. Nos.4,816,567; and 4,816,397, which are incorporated herein by reference intheir entirety.) Humanized antibodies are antibody molecules fromnon-human species having one or more complementarity determining regions(CDRs) from the non-human species and a framework region from a humanimmunoglobulin molecule. (See, e.g., U.S. Pat. No. 5,585,089, which isincorporated herein by reference in its entirety.) Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inInternational Publication No. WO 87/02671; European Patent PublicationNo. 0 184 187; European Patent Publication No. 0 171 496; EuropeanPatent Publication No. 0 173 494; International Publication No. WO86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No. 012023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc.Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985,Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986,BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler etal., 1988, J. Immunol. 141:4053-4060; each of which is incorporatedherein by reference in its entirety.

Completely human antibodies are particularly desirable and can beproduced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, 1995, Int. Rev.Immunol. 13:65-93. For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which isincorporated herein by reference in its entirety. Other human antibodiescan be obtained commercially from, for example, Abgenix, Inc. (nowAmgen, Freemont, Calif.) and Medarex (Princeton, N.J.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (See, e.g., Jespers et al., 1994,Biotechnology 12:899-903). Human antibodies can also be produced usingvarious techniques known in the art, including phage display libraries(see, e.g., Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks etal., 1991, J. Mol. Biol. 222:581; Quan and Carter, 2002, The rise ofmonoclonal antibodies as therapeutics, In Anti-IgE and Allergic Disease,Jardieu and Fick, eds., Marcel Dekker, New York, N.Y., Chapter 20, pp.427-469).

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, preferably at least 10, 20 or 50 amino acidportion of the protein) that is not from an antibody. Preferably, theantibody or fragment thereof is covalently linked to the other proteinat the N-terminus of the constant domain.

Antibodies include analogs and derivatives that are either modified,i.e., by the covalent attachment of any type of molecule as long as suchcovalent attachment permits the antibody to retain its antigen bindingimmunospecificity. For example, but not by way of limitation,derivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

Antibodies can have modifications (e.g., substitutions, deletions oradditions) in amino acid residues that interact with Fc receptors. Inparticular, antibodies can have modifications in amino acid residuesidentified as involved in the interaction between the anti-Fc domain andthe FcRn receptor (see, e.g., International Publication No. WO 97/34631,which is incorporated herein by reference in its entirety).

Antibodies immunospecific for a cancer cell antigen can be obtainedcommercially or produced by any method known to one of skill in the artsuch as, e.g., chemical synthesis or recombinant expression techniques.The nucleotide sequence encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, literature publications, or by routine cloning andsequencing.

In a specific embodiment, known antibodies for the treatment of cancercan be used. Antibodies immunospecific for a cancer cell antigen can beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., recombinant expression techniques. The nucleotidesequence encoding antibodies immunospecific for a cancer cell antigencan be obtained, e.g., from the GenBank database or a database like it,the literature publications, or by routine cloning and sequencing.Examples of antibodies available for the treatment of cancer include,but are not limited to, OVAREX which is a murine antibody for thetreatment of ovarian cancer; PANOREX (Glaxo Wellcome, N.C.) which is amurine IgG_(2a) antibody for the treatment of colorectal cancer;Cetuximab ERBITUX (Imclone Systems Inc., NY) which is an anti-EGFR IgGchimeric antibody for the treatment of epidermal growth factor positivecancers, such as head and neck cancer; Vitaxin (MedImmune, Inc., MD)which is a humanized antibody for the treatment of sarcoma; CAMPATH I/H(Leukosite, MA) which is a humanized IgG₁ antibody for the treatment ofchronic lymphocytic leukemia (CLL); SMART ID10 (Protein Design Labs,Inc., CA) which is a humanized anti-HLA-DR antibody for the treatment ofnon-Hodgkin's lymphoma; ONCOLYM (Techniclone, Inc., CA) which is aradiolabeled murine anti-HLA-Dr10 antibody for the treatment ofnon-Hodgkin's lymphoma; ALLOMUNE (BioTransplant, CA) which is ahumanized anti-CD2 mAb for the treatment of Hodgkin's Disease ornon-Hodgkin's lymphoma; and CEACIDE (Immunomedics, NJ) which is ahumanized anti-CEA antibody for the treatment of colorectal cancer.

In attempts to discover effective cellular targets for cancer diagnosisand therapy, researchers have sought to identify transmembrane orotherwise tumor-associated polypeptides that are specifically expressedon the surface of one or more particular type(s) of cancer cell ascompared to on one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

The Linker Unit (L)

A linker (sometimes referred to as “[linker]” herein) is a bifunctionalcompound which can be used to link a drug and an antibody to form anantibody drug conjugate (ADC). Such conjugates are useful, for example,in the formation of immunoconjugates directed against tumor associatedantigens. Such conjugates allow the selective delivery of cytotoxicdrugs to tumor cells.

In an ADC the linker serves to attach the payload to the antibody.

In one aspect, a second section of the linker unit is introduced whichhas a second reactive site e.g., an electrophilic group that is reactiveto a nucleophilic group present on an antibody unit (e.g., an antibody).Useful nucleophilic groups on an antibody include but are not limitedto, sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of an antibody is reactive to an electrophilic groupon a linker unit and forms a covalent bond to a linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups. The electrophilic group provides a convenient sitefor antibody attachment.

In another embodiment, a linker unit has a reactive site which has anucleophilic group that is reactive to an electrophilic group present onan antibody. Useful electrophilic groups on an antibody include, but arenot limited to, aldehyde and ketone carbonyl groups. The heteroatom of anucleophilic group of a linker unit can react with an electrophilicgroup on an antibody and form a covalent bond to the antibody. Usefulnucleophilic groups on a linker unit include, but are not limited to,hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide. The electrophilic group on an antibodyprovides a convenient site for attachment to a linker unit.

Amino functional groups are also useful reactive sites for a linker unitbecause they can react with carboxylic acid, or activated esters of acompound to form an amide linkage. Typically, the peptide-basedcompounds of the invention can be prepared by forming a peptide bondbetween two or more amino acids and/or peptide fragments. Such peptidebonds can be prepared, for example, according to the liquid phasesynthesis method (see, e.g., Schroder and Lubke, “The Peptides”, volume1, pp 76-136, 1965, Academic Press) that is well known in the field ofpeptide chemistry.

In the context of the invention, particularly but not limited to linkercomponents such as L₁, L₂ (including L₂ ^(A), L₂ ^(B) and L₂ ^(C)) andL₃, the language “selected from one or more of” or “one or more of”indicates that multiple components, which may be the same or different,are or may be arranged sequentially. Thus, for example, L₃ may be—C₁₋₆alkyl-, —NR— or the other individually listed components, but also—C₁₋₆alkyl-NR—, or any other combination of 2 or more listed components.

In another embodiment, a linker unit has a reactive site that can reactwith antibody nucleophiles, such as cysteins. The reactive site iscomprised of a heterocycle that is substituted with a sulfone. Thesulfone is then replaced by the antibody nucleophile (i.e. cysteine) andthe newly formed bond between the antibody and the heterocycle connectsthe antibody to the linker. See, WO 2014/144878.

Synthesis of Compounds and Antibody Drug Conjugates Thereof

The compounds and conjugates of the invention can be made using thesynthetic procedures outlined below in the Exemplification. As describedin more detail below, the compounds and conjugates of the invention canbe prepared using a section of a linker unit having a reactive site forbinding to the compound. In one aspect, a second section of the linkerunit is introduced which has a second reactive site e.g., anelectrophilic group that is reactive to a nucleophilic group present onan antibody unit (e.g., an antibody). Useful nucleophilic groups on anantibody include but are not limited to, sulfhydryl, hydroxyl and aminogroups. The heteroatom of the nucleophilic group of an antibody isreactive to an electrophilic group on a linker unit and forms a covalentbond to a linker unit. Useful electrophilic groups include, but are notlimited to, maleimide and haloacetamide groups. The electrophilic groupprovides a convenient site for antibody attachment.

In another embodiment, a linker unit has a reactive site which has anucleophilic group that is reactive to an electrophilic group present onan antibody. Useful electrophilic groups on an antibody include, but arenot limited to, aldehyde and ketone carbonyl groups. The heteroatom of anucleophilic group of a linker unit can react with an electrophilicgroup on an antibody and form a covalent bond to the antibody. Usefulnucleophilic groups on a linker unit include, but are not limited to,hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide. The electrophilic group on an antibodyprovides a convenient site for attachment to a linker unit.

Amino functional groups are also useful reactive sites for a linker unitbecause they can react with carboxylic acid, or activated esters of acompound to form an amide linkage. Typically, the peptide-basedcompounds of the invention can be prepared by forming a peptide bondbetween two or more amino acids and/or peptide fragments. Such peptidebonds can be prepared, for example, according to the liquid phasesynthesis method (see, e.g., Schroder and Lubke, “The Peptides”, volume1, pp 76-136, 1965, Academic Press) that is well known in the field ofpeptide chemistry.

As described in more detail below, the conjugates can be prepared usinga section of the linker having a reactive site for binding to a compoundof the invention and introducing another section of the linker unithaving a reactive site for an antibody. In one aspect, a linker unit hasa reactive site which has an electrophilic group that is reactive with anucleophilic group present on an antibody unit, such as an antibody. Theelectrophilic group provides a convenient site for antibody attachment.Useful nucleophilic groups on an antibody include but are not limitedto, sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of an antibody is reactive to an electrophilic groupon a linker unit and forms a covalent bond to a linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups.

In another embodiment, a linker unit has a reactive site which has anucleophilic group that is reactive with an electrophilic group presenton an antibody unit. The electrophilic group on an antibody provides aconvenient site for attachment to a linker unit. Useful electrophilicgroups on an antibody include, but are not limited to, aldehyde andketone carbonyl groups. The heteroatom of a nucleophilic group of alinker unit can react with an electrophilic group on an antibody andform a covalent bond to the antibody. Useful nucleophilic groups on alinker unit include, but are not limited to, hydrazide, oxime, amino,hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

In another embodiment of the invention the linkage of the presentinvention employs an engineered antibody constant domain polypeptide, ora portion thereof, wherein the engineered constant domain comprises atleast one amino acid substitution to introduce a cysteine residue usefulfor conjugation, and wherein the constant domain polypeptide is anengineered human IgG heavy chain constant domain (Cy) polypeptide, orportion thereof, comprising at least one amino acid substitutionselected from the group consisting of at K246, D249, D265, S267, D270,N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, N315, E318,K320, 1332, E333, K334, 1336, E345, Q347, S354, R355, M358, K360, Q362,K370, Y373, D376, A378, E380, E382, Q386, E388, N390, K392, T393, D401,F404, T411, D413, K414, R416, Q418, Q419, N421, M428, A431, L432, T437,Q438, K439, L443, and S444, according to the EU index of Kabat.

In another embodiment of the invention the linkage of the presentinvention employs an engineered antibody constant domain polypeptide, ora portion thereof, wherein the engineered constant domain comprises atleast one amino acid substitution to introduce a cysteine residue usefulfor conjugation, and wherein the constant domain polypeptide is anengineered human lambda light chain constant domain (CA) polypeptide, orportion thereof, comprising at least one amino acid substitutionselected from the group consisting of K110, A111, L125, K149C, V155,G158, T161, Q185, S188, H189, S191, T197, V205, E206, K207, T208 andA210, according to the numbering of Kabat.

In yet another embodiment of the invention the linkage of the presentinvention employs an engineered antibody constant domain polypeptide, ora portion thereof, wherein the engineered constant domain comprises atleast one amino acid substitution to introduce a cysteine residue usefulfor conjugation, and wherein the constant domain polypeptide is anengineered human kappa light chain constant domain (CK) polypeptide, orportion thereof, comprising at least one amino acid substitutionselected from the group consisting of A111, K183, and N210, according tothe numbering of Kabat.

In yet another embodiment of the invention the linkage of the presentinvention employs an engineered antibody constant domain polypeptide, ora portion thereof, wherein the engineered constant domain comprises atleast one amino acid substitution to introduce a cysteine residue usefulfor conjugation, and wherein the constant domain polypeptide is anengineered Cy polypeptide, or portion thereof, comprising at least oneamino acid sequence selected from the group consisting of an amino acidsequence of SEQ ID NOs:97-100, 102, 104, 107-127, and 129-163, asdisclosed in WO2013/093809, which reference is incorporated herein inits entirety.

In still another embodiment of the invention the linkage of the presentinvention employs an engineered antibody constant domain polypeptide, ora portion thereof, wherein the engineered constant domain comprises atleast one amino acid substitution to introduce a cysteine residue usefulfor conjugation, and wherein the constant domain polypeptide is anengineered CK polypeptide, or portion thereof, comprising at least oneamino acid sequence selected from the group consisting of an amino acidsequence of SEQ ID NOs:90, 92, 95, 164, 166, and 169, as disclosed inWO2013/093809, which reference is incorporated herein in its entirety.

In still another embodiment of the invention the linkage of the presentinvention employs an engineered antibody constant domain polypeptide, ora portion thereof, wherein the engineered constant domain comprises atleast one amino acid substitution to introduce a cysteine residue usefulfor conjugation, and wherein the constant domain polypeptide is anengineered CA polypeptide, or portion thereof, comprising at least oneamino acid sequence selected from the group consisting of an amino acidsequence of SEQ ID NOs:172-186, as disclosed in WO2013/093809, whichreference is incorporated herein in its entirety.

In another embodiment of the invention the engineered Cy polypeptidementioned above further comprises at least one mutation selected fromthe group consisting of a mutation at amino acid position 284, 287,A327, N384, L³⁹⁸, and V⁴²², according to the EU index of Kabat.

Conjugation with Transglutaminase

In certain embodiments, a compound of the invention may be covalentlycrosslinked to an Fc-containing or Fab-containing polypeptide engineeredwith an acyl donor glutamine-containing tag (e.g., Gln-containingpeptide tags or Q-tags) or an endogenous glutamine made reactive (i.e.,the ability to form a covalent bond as an acyl donor in the presence ofan amine and a transglutaminase) by polypeptide engineering (e.g., viaamino acid deletion, insertion, substitution, mutation, or anycombination thereof on the polypeptide), in the presence oftransglutaminase, provided that the compound of the invention comprisesan amine donor agent (e.g., small molecule comprising or attached to areactive amine), thereby forming a stable and homogenous population ofan engineered Fc-containing polypeptide conjugate with the amine donoragent being site-specifically conjugated to the Fc-containing orFab-containing polypeptide through the acyl donor glutamine-containingtag or the exposed/accessible/reactive endogenous glutamine. Forexample, compounds of the invention may be conjugated as described inInternational Patent Application Serial No. PCT/IB2011/054899, whoseentire contents are incorporated herein by reference. In certainembodiments, to facilitate conjugation of the compound of the inventionto an Fc-containing or Fab-containing polypeptide engineered with anacyl donor glutamine-containing tag or an endogenous glutamine madereactive by polypeptide engineering in the presence of transglutaminase,Z is NH₂.

Conjugation to the Human Light Chain Kappa Domain Constant Region

In certain embodiments, a compound of the invention may be covalentlyattached to the side chain of K¹⁸⁸ of the human light chain kappa domainconstant region (CLκ) (full light chain numbering according to Kabat).For example, compounds of the invention may be conjugated as describedin U.S. patent application Ser. No. 13/180,204, whose entire contentsare incorporated herein by reference. In certain embodiments, tofacilitate conjugation to K188 CLκ, Z is

R⁷ is independently selected for each occurrence from the groupconsisting of F, Cl, I, Br, NO₂, CN and CF₃; and h is 1, 2, 3, 4 or 5.

In certain embodiments, the invention provides for a compositioncomprising a compound of the invention covalently conjugated to anantibody (or antigen binding portion thereof), wherein at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90% of the compound of the invention in thecomposition is conjugated to the antibody or antigen binding portionthereof at K¹⁸⁸ CLκ.

In certain embodiments, the compounds of the invention may be conjugatedto the combining site of a catalytic antibody, such as aldolaseantibodies, or antigen binding portion thereof. Aldolase antibodiescontain combining site portions that, when unencumbered (for example byconjugation), catalyze an aldol addition reaction between an aliphaticketone donor and an aldehyde acceptor. The contents of US PatentApplication Publication No. US 2006/205670 are incorporated herein byreference, in particular pages 78-118 describing linkers, and paragraphs[0153]-[0233] describing antibodies, useful fragments, variants andmodifications thereof, h38C2, combining sites and complimentarydetermining regions (CDRs), and related antibody technology. The term“combining site” includes the CDRs and the adjacent framework residuesthat are involved in antigen binding.

Compositions and Methods of Administration

In other embodiments, another aspect of the invention relates topharmaceutical compositions including an effective amount of a compoundof the invention and/or antibody drug conjugate thereof and apharmaceutically acceptable carrier or vehicle. In certain embodiments,the compositions are suitable for veterinary or human administration.

The present pharmaceutical compositions can be in any form that allowsfor the composition to be administered to a patient. For example, thecomposition can be in the form of a solid or liquid. Typical routes ofadministration include, without limitation, parenteral, ocular andintra-tumor. Parenteral administration includes subcutaneous injections,intravenous, intramuscular or intrasternal injection or infusiontechniques. In one aspect, the compositions are administeredparenterally. In a specific embodiment, the compositions areadministered intravenously.

Pharmaceutical compositions can be formulated so as to allow a compoundof the invention and/or antibody drug conjugate thereof to bebioavailable upon administration of the composition to a patient.Compositions can take the form of one or more dosage units, where forexample, a tablet can be a single dosage unit, and a container of acompound of the invention and/or antibody drug conjugate thereof inliquid form can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the a compound of the invention and/orantibody drug conjugate thereof, the manner of administration, and thecomposition employed.

The pharmaceutically acceptable carrier or vehicle can be solid orparticulate, so that the compositions are, for example, in tablet orpowder form. The carrier(s) can be liquid. In addition, the carrier(s)can be particulate.

The composition can be in the form of a liquid, e.g., a solution,emulsion or suspension. In a composition for administration byinjection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates,phosphates or amino acids and agents for the adjustment of tonicity suchas sodium chloride or dextrose. A parenteral composition can be enclosedin ampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of a compound of the invention and/or antibody drug conjugatethereof that is effective in the treatment of a particular disorder orcondition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. In addition, in vitroor in vivo assays can optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the compositions willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances.

The compositions comprise an effective amount of a compound of theinvention and/or antibody drug conjugate thereof such that a suitabledosage will be obtained. Typically, this amount is at least about 0.01%of a compound of the invention and/or antibody drug conjugate thereof byweight of the composition. In an exemplary embodiment, pharmaceuticalcompositions are prepared so that a parenteral dosage unit contains fromabout 0.01% to about 2% by weight of the amount of a compound of theinvention and/or antibody drug conjugate thereof.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of a compound of the invention and/or antibody drugconjugate thereof per kg of the patient's body weight. In one aspect,the composition can include from about 1 to about 100 mg of a compoundof the invention and/or antibody drug conjugate thereof per kg of thepatient's body weight. In another aspect, the amount administered willbe in the range from about 0.1 to about 25 mg/kg of body weight of acompound of the invention and/or antibody drug conjugate thereof.

Generally, the dosage of a compound of the invention and/or antibodydrug conjugate thereof administered to a patient is typically about 0.01mg/kg to about 20 mg/kg of the patient's body weight. In one aspect, thedosage administered to a patient is between about 0.01 mg/kg to about 10mg/kg of the patient's body weight. In another aspect, the dosageadministered to a patient is between about 0.1 mg/kg and about 10 mg/kgof the patient's body weight. In yet another aspect, the dosageadministered to a patient is between about 0.1 mg/kg and about 5 mg/kgof the patient's body weight. In yet another aspect the dosageadministered is between about 0.1 mg/kg to about 3 mg/kg of thepatient's body weight. In yet another aspect, the dosage administered isbetween about 1 mg/kg to about 3 mg/kg of the patient's body weight.

A compound of the invention and/or antibody drug conjugate thereof canbe administered by any convenient route, for example by infusion orbolus injection. Administration can be systemic or local. Variousdelivery systems are known, e.g., encapsulation in liposomes,mieroparticles, microcapsules, capsules, etc., and can be used toadminister a compound of the invention and/or antibody drug conjugatethereof. In certain embodiments, more than one compound of the inventionand/or antibody drug conjugate thereof is administered to a patient.

In specific embodiments, it can be desirable to administer one or morecompounds of the invention and/or antibody drug conjugates thereoflocally to the area in need of treatment. This can be achieved, forexample, and not by way of limitation, by local infusion during surgery;topical application, e.g., in conjunction with a wound dressing aftersurgery; by injection; by means of a catheter; or by means of animplant, the implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.In one embodiment, administration can be by direct injection at the site(or former site) of a cancer, tumor or neoplastic or pre-neoplastictissue. In another embodiment, administration can be by direct injectionat the site (or former site) of a manifestation of an autoimmunedisease.

In yet another embodiment, the compound of the invention and/or antibodydrug conjugate thereof can be delivered in a controlled release system,such as but not limited to, a pump or various polymeric materials can beused. In yet another embodiment, a controlled-release system can beplaced in proximity of the target of the compound of the inventionand/or antibody drug conjugate thereof, e.g., the liver, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer(Science 249:1527-1533 (1990)) can be used.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a compound or antibody drug conjugate thereof is administered.Such pharmaceutical carriers can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin. Thecarriers can be saline, and the like. In addition, auxiliary,stabilizing and other agents can be used. In one embodiment, whenadministered to a patient, the compound or conjugate andpharmaceutically acceptable carriers are sterile. Water is an exemplarycarrier when the compound or conjugate are administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions. Thepresent compositions, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents.

The present compositions can take the form of solutions, pellets,powders, sustained-release formulations, or any other form suitable foruse. Other examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

In an embodiment, the compound of the invention and/or antibody drugconjugate thereof are formulated in accordance with routine proceduresas a pharmaceutical composition adapted for intravenous administrationto animals, particularly human beings. Typically, the carriers orvehicles for intravenous administration are sterile isotonic aqueousbuffer solutions. Where necessary, the compositions can also include asolubilizing agent. Compositions for intravenous administration canoptionally comprise a local anesthetic such as lignocaine to ease painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where a compound of the invention and/or antibody drugconjugate thereof is to be administered by infusion, it can bedispensed, for example, with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the compound of theinvention and/or antibody drug conjugate thereof is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients can be mixed prior to administration.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

Whether in solid or liquid form, the present compositions can include apharmacological agent used in the treatment of cancer.

Therapeutics Uses of Compounds and Antibody Drug Conjugates Thereof

Another aspect of the invention relates to a method of using thecompounds of the invention and antibody drug conjugates thereof fortreating cancer.

The compounds of the invention and/or antibody drug conjugates thereofare useful for inhibiting the multiplication of a tumor cell or cancercell, causing apoptosis in a tumor or cancer cell, or for treatingcancer in a patient. The compounds of the invention and/or antibody drugconjugates thereof can be used accordingly in a variety of settings forthe treatment of animal cancers. Said conjugates can be used to delivera compound of the invention to a tumor cell or cancer cell. Withoutbeing bound by theory, in one embodiment, the antibody of the conjugatebinds to or associates with a cancer-cell or a tumor-cell-associatedantigen, and the conjugate can be taken up (internalized) inside a tumorcell or cancer cell through receptor-mediated endocytosis or otherinternalization mechanism. The antigen can be attached to a tumor cellor cancer cell or can be an extracellular matrix protein associated withthe tumor cell or cancer cell. In certain embodiments, once inside thecell, one or more specific peptide sequences are enzymatically orhydrolytically cleaved by one or more tumor cell or cancercell-associated proteases, resulting in release of a compound of theinvention from the conjugate. The released compound of the invention isthen free to migrate within the cell and induce cytotoxic or cytostaticactivities. The conjugate also can be cleaved by an intracellularprotease to release a compound of the invention. In an alternativeembodiment, the compound of the invention is cleaved from conjugateoutside the tumor cell or cancer cell, and the compound of the inventionsubsequently penetrates the cell.

In certain embodiments, the conjugates provide conjugation-specifictumor or cancer drug targeting, thus reducing general toxicity of thecompounds of the invention.

In another embodiment, the antibody unit binds to the tumor cell orcancer cell.

In another embodiment, the antibody unit binds to a tumor cell or cancercell antigen which is on the surface of the tumor cell or cancer cell.

In another embodiment, the antibody unit binds to a tumor cell or cancercell antigen which is an extracellular matrix protein associated withthe tumor cell or cancer cell.

The specificity of the antibody unit for a particular tumor cell orcancer cell can be important for determining those tumors or cancersthat are most effectively treated.

Particular types of cancers that can be treated with a compound of theinvention and/or antibody drug conjugate thereof, include but are notlimited to, carcinomas of the bladder, breast, cervix, colon,endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, skin,stomach, and testes; and blood born cancers including but not limited toleukemias and lymphomas.

Multi-Modality Therapy for Cancer. Cancers, including, but not limitedto, a tumor, metastasis, or other disease or disorder characterized byuncontrolled cell growth, can be treated or inhibited by administrationof a compound of the invention and/or antibody drug conjugate thereof.

In other embodiments, methods for treating cancer are provided,including administering to a patient in need thereof an effective amountof a compound of the invention and/or antibody drug conjugate thereofand a chemotherapeutic agent. In one embodiment the chemotherapeuticagent is that with which treatment of the cancer has not been found tobe refractory. In another embodiment, the chemotherapeutic agent is thatwith which the treatment of cancer has been found to be refractory. Acompound of the invention and/or antibody drug conjugate thereof can beadministered to a patient that has also undergone surgery as treatmentfor the cancer.

In some embodiments, the patient also receives an additional treatment,such as radiation therapy. In a specific embodiment, the compound of theinvention and/or antibody drug conjugate thereof is administeredconcurrently with the chemotherapeutic agent or with radiation therapy.In another specific embodiment, the chemotherapeutic agent or radiationtherapy is administered prior or subsequent to administration of acompound of the invention and/or antibody drug conjugate thereof.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents, such a standardof care chemotherapeutic agent(s), can be administered.

Additionally, methods of treatment of cancer with a compound of theinvention and/or antibody drug conjugate thereof are provided as analternative to chemotherapy or radiation therapy where the chemotherapyor the radiation therapy has proven or can prove too toxic, e.g.,results in unacceptable or unbearable side effects, for the subjectbeing treated. The patient being treated can, optionally, be treatedwith another cancer treatment such as surgery, radiation therapy orchemotherapy, depending on which treatment is found to be acceptable orbearable.

The compounds of the invention and/or antibody drug conjugates thereofcan also be used in an in vitro or ex vivo fashion, such as for thetreatment of certain cancers, including, but not limited to leukemiasand lymphomas, such treatment involving autologous stem celltransplants. This can involve a multi-step process in which the animal'sautologous hematopoietic stein cells are harvested and purged of allcancer cells, the animal's remaining bone-marrow cell population is theneradicated via the administration of a high dose of a compound of theinvention and/or antibody drug conjugate thereof with or withoutaccompanying high dose radiation therapy, and the stem cell graft isinfused back into the animal. Supportive care is then provided whilebone marrow function is restored and the patient recovers.

The invention is further described in the following examples, which arein not intended to limit the scope of the invention.

General Methods

Synthetic Experimental Procedures:

Experiments were generally carried out under inert atmosphere (nitrogenor argon), particularly in cases where oxygen- or moisture-sensitivereagents or intermediates were employed. Commercial solvents andreagents were generally used without further purification, includinganhydrous solvents where appropriate (generally Sure-Sea™ products fromthe Aldrich Chemical Company, Milwaukee, Wis.). Mass spectrometry datais reported from either liquid chromatography-mass spectrometry (LC-MS)or atmospheric pressure chemical ionization (APCI). Chemical shifts fornuclear magnetic resonance (NMR) data are expressed in parts per million(ppm, 6) referenced to residual peaks from the deuterated solventsemployed.

For syntheses referencing procedures in other Examples or Methods,reaction Protocol (length of reaction and temperature) may vary. Ingeneral, reactions were followed by thin layer chromatography, LC-MS orHPLC, and subjected to work-up when appropriate. Purifications may varybetween experiments: in general, solvents and the solvent ratios usedfor eluents/gradients were chosen to provide appropriate retentiontimes. Unless otherwise specified, reverse phase HPLC fractions wereconcentrated via lyophilization/Freeze-drying.

Intermediate and final compounds were stored at (0° C.) or roomtemperature in closed vials or flasks under nitrogen. Compound nameswere generated with ACD Labs software.

Abbreviations for solvents and/or reagents is based on American ChemicalSociety guidelines and is highlighted below:

Ac=Acetyl

Boc=N-tert-butoxycarbonyl

CDI=N,N′-Carbonyldiimidazole

DCC=1,3-Dicyclohexylcarbodiimide

DCE=Dichloroethane

DCM=Dichloromethane

DEA=N,N-Diethylamine

DEAD=Diethyl azodicarboxylate

DIAD=Diisopropyl azodicarboxylate

DIBAL-H=Diisobutylaluminium hydride

DIPEA (or) Hunig's Base=N,N-Diisopropylethylamine

DMA=Dimethylacetamide

DMAP=4-Dimethylaminopyridine

DME=Dimethoxyethane

DMF=N,N-Dimethylformamide

DMSO=Dimethyl sulfoxide

DPPA=Diphenylphosphoryl azide

EDCI=1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

EtOAc=Ethyl acetate

Fmoc=Fluorenylmethyloxycarbonyl

h=hour

HATU=o-(7-azabenzotriazol-1-yl)-N,N,N′,N′-te-tramethyluroniumhexafluorophosphate

HBTU=N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate

HOAc=Acetic acid

HOAt=1-Hydroxy-7-azabenzotriazole

HOBt=1-Hydroxybenzotriazole hydrate

LDA=Lithium diisopropylamide

Me=Methyl

MS=Molecular Sieves

MTBE=Methyl tert-butyl ether

n-BuLi=n-Butyllithium

NBS=N-Bromosuccinimide

NMM=N-methyl morpholine

Ph=Phenyl

PPTS=Pyridinium p-Toluenesulfonate

p-TsOH=p-Toluenesulfonic acid

rt=room temperature

TBAI=Tetrabutylammonium Iodide

TEA=Triethylamine

Tf=Trifluoromethanesulfonate

TFA=TFA

THF=Tetrahydrofuran

TPTU=O-(2-Oxo-1(2H)pyridyl)-N,N,N,′N′-tetramethyluroniumtetrafluoroborate

HPLC and LC-MS Conditions Used for Analysis

Protocol A: Column: Waters Acquity UPLC HSS T3, 2.1 mm×50 mm, C18, 1.7μm; Mobile phase A:0.1% formic acid in water (v/v); Mobile phase B: 0.1%formic acid in acetonitrile (v/v); Gradient: 5% B over 0.1 minute, 5% to95% B over 2.5 minutes, 95% B over 0.35 minute; Flow rate: 1.25mL/minute. Temperature: 60° C.; Detection: 200-450 nm; MS (+) range100-2000 daltons; Injection volume: 5 μL; Instrument: Waters Acquity.

Protocol B: Column: Waters Acquity UPLC HSS T3, C18, 2.1×50 mm, 1.7 μm;Mobile phase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1%formic acid in acetonitrile (v/v); Gradient: 5% B over 0.1 minute, 5% to95% B over 1.5 minute, 95% B over 0.35 minute; Flow rate: 1.25mL/minute. Temperature: 60° C.; Detection: 200-450 nm; MS (+) range100-2000 daltons; Injection volume: 5 μL; Instrument: Waters Acquity.

HPLC Conditions Used for Purification

Method A: Column: Phenomenex Luna Phenylhexyl 150×21.2 mm, 5 μm; Mobilephase A: 0.02% TFA in water (v/v); Mobile phase B: 0.02% TFA inacetonitrile (v/v); Gradient: 20% B over 1.5 minutes, 20% B to 100% Bover 8.5 minutes, then 100% B over 2.0 minutes; Flow rate: 27 mL/minute.Temperature: not controlled; Detection: DAD 210-360 nm; MS (+) range150-2000 daltons; Instrument: 305 RP Waters Fractional Lynx LCMS

Method B: Column: Phenomenex Luna C18, 100×30 mm, 5 μm; Mobile phase A:0.02% TFA in water (v/v); Mobile phase B: 0.02% TFA in acetonitrile(v/v); Gradient: 20% B over 1.5 minutes, 20% B to 100% B over 8.5minutes, then 95% B over 2.0 minutes; Flow rate: 30 mL/minute.Temperature: not controlled; Detection: UV 215 nm; Instrument: Gilson

Method C: Phenomenex Luna C18, 100×30 mm, 5 μm; Mobile phase A: 0.02%TFA in water (v/v); Mobile phase B: 0.02% TFA in acetonitrile (v/v);Gradient: variable, increasing gradient of B in A over 10 to 20 minutes.Flow rate: 27 to 30 mL/minute. Temperature: not controlled; Detection:UV 215 nm; Instrument: Gilson

General Procedures:

General procedure A: To a stirring solution of the mono or diacid, inTHF, dichloromethane, or a mixture of both at 0° C., oxalyl chloride(1-2.5 eq.) was added followed by a catalytic amount of DMF. Thereaction allowed to stir at 0° C. for several minutes before beingallowed to warm to room temperature, and then stir at room temperaturefor 30 minutes to several hours. The reaction was then concentrated invacuo. In some cases the crude material was then azeotroped one toseveral times with heptane, or other relevant solvent or solvents. Crudematerial was then dried over high vacuum before being used in the nextstep.

General procedure B: To a stirring solution of the amine (2-2.5 eq.) inTHF, dichloromethane, or a mixture of both at 0° C. (or in some casesother relevant solvent or solvents), the acid chloride, or diacidchloride was added followed by pyridine (3-6 eq.), triethylamine (3-6eq.), or other relevant base (3-6 eq.). The reaction allowed to stir at0° C. for a few seconds to several minutes before being allowed to warmto room temperature, and then stir at room temperature for 10 minutes toseveral hours. The reaction was then concentrated in vacuo. In somecases the crude material was then azeotroped one to several times withheptane, or other relevant solvent or solvents. In most cases the crudematerial was then purified by a described method such as silicachromatography or medium pressure reverse phase C18 chromatography.

Examples Preparation of(S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylAcetate (4)

Step 1: Synthesis of tert-butyl(S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(2). To a stirring solution of tert-butyl(8S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(1) [prepared as described in J. Am. Chem. Soc., 2007, 129,14092-14099.] (100 mg, 0.225 mmol) in THF (1.5 mL) was added 10% Pd/C(33 mg) followed by a 25% aqueous solution of ammonium formate (0.15mL). The solution was stirred for 2 h. The solution was diluted withether (6 mL) and sodium sulfate was added. The mixture was filteredthrough Celite and solvent removed in vacuo providing the desiredproduct 2 as a white solid (79 mg, 100%).

Step 2: Synthesis of tert-butyl(S)-4-acetoxy-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(3). tert-Butyl(S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(2.69 mg, 0.19 mmol) was dissolved in CH₂Cl₂ (2 ml). Pyridine (46 mg,0.585 mmol). Acetyl chloride (16 mL, 0.234 mmol) was added and reactionstirred for 2 h. The solvent was removed in vacuo. The crude residue waspurified by flash chromatography (0-100% EtOAc/heptane) to providedesired product (45 mg, 58%).

Step 3: Synthesis of(S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylacetate (4): tert-butyl(S)-4-acetoxy-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(3) (45 mg, 0.11 mmol) was taken up in dioxane (1 mL). 4N HCl in dioxane(1 mL) was added and solution was stirred for 2 h. Solvent was removedto provide crude target material in quantitative yield and the materialwas used immediately as is.

Preparation of Bicyclo[1.1.1]-1,3-dicarboxylic Acid Chloride (6)

Bicyclo[1.1.1]pentane-1,3-dicarboxylic acid (50 mg) was placed in vialand taken up in THF (1 mL) and a drop of DMF was added. Oxalyldichloride (122 mg, 0.961 mmol, 0.0825 mL) was added slowly and solutionbubbled rapidly. After 2 h the solvent was removed and the diacidchloride 6 was used as is in subsequent reactions.

Preparation of(8S,8'S)-(bicyclo[1.1.1]pentane-1,3-dicarbonyl)bis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)Diacetate (7)

Step 1:(8S,8'S)-(bicyclo[1.1.1]pentane-1,3-dicarbonyl)bis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)Diacetate (7): The title compound was prepared following generalprocedure B using 4 (20.0 mg, 0.060 mmol), 6 (5.81 mg, 0.0301 mmol,0.0301 mL, 1 M in THF), pyridine (14.3 0.181 mmol) and THF (1.0 mL), andpurification using Material was purified by medium pressure reversephase C18 chromatography (Gradient: 0% to 80% acetonitrile in water with0.02% TFA in each phase) (providing desired product 7. (8.0 mg, 20%).LC-MS (Protocol B): m/z 711.3 [M+H]⁺, retention time=1.12 minutes

Preparation of(R)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylAcetate (11)

Step 1: Synthesis of tert-butyl(R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(9). To a stirring solution of tert-butyl(8R)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(8) [prepared as described in J. Am. Chem. Soc., 2007, 129,14092-14099.] (100 mg, 0.225 mmol) in THF (1.5 mL) was added 10% Pd/C(33 mg) followed by a 25% aqueous solution of ammonium formate (0.15mL). The solution was stirred for 2 h. The solution was diluted withether (6 mL) and sodium sulfate was added. The mixture was filteredthrough Celite and solvent removed in vacuo providing the desiredproduct 9 as a white solid (79 mg, 100%).

Step 2: Synthesis of tert-butyl(R)-4-acetoxy-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(10): tert-Butyl(R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(2) (69 mg, 0.19 mmol) was dissolved in CH₂Cl₂ (2 ml). pyridine (46 mg,0.585 mmol) was added. Acetyl chloride (16 mL, 0.234 mmol) was added andreaction stirred for 2 h. The solvent was removed in vacuo. The cruderesidue was purified by flash chromatography (0-100% EtOAc/heptane) toprovide desired product 10 (53 mg, 69%).

Step 3: Synthesis of(R)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylacetate (11): tert-butyl(R)-4-acetoxy-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(10) (53 mg, 0.13 mmol) was taken up in dioxane (1 mL). 4N HCl indioxane (1 mL) was added and solution was stirred for 2 h. Solvent wasremoved to provide crude target material in quantitative yield and thematerial was used immediately as is Preparation of(8R,8′R)-(bicyclo[1.1.1]pentane-1,3-dicarbonyl)bis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)diacetate (11).

Preparation of(8R,8′R)-(bicyclo[1.1.1]pentane-1,3-dicarbonyl)bis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)Diacetate (12)

Step 1: Synthesis of(8R,8′R)-(bicyclo[1.1.1]pentane-1,3-dicarbonyl)bis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)diacetate (12).

The title compound was prepared following general procedure B using11(20.0 mg, 0.060 mmol) and 6 (5.81 mg, 0.0301 mmol, 0.0301 mL, 1 M inTHF), pyridine (14.3 0.181 mmol) and THE (1.0 mL), and purificationusing medium pressure reverse phase C₁₈ chromatography (Gradient: 0% to80% acetonitrile in water with 0.02% TFA in each phase) providingdesired product 12. (5.9 mg, 14%). LC-MS (Protocol B): m/z 711.2 [M+H]⁺,retention time=1.12 minutes

Preparation of(S)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indol-4-ylAcetate (16)

Step 1: Synthesis of tert-butyl(S)-8-(chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(14): tert-Butyl(S)-4-(benzyloxy)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(13) [prepared as described in J. Am. Chem. Soc., 2007, 129,14092-14099.] (250 mg, 0.563 mmol) was taken up in THF (5.63 mL). 10%Pd/C (80 mg) was added. A freshly prepared 25% aqueous Formic acidammonium salt (500 mg, 2 mmol, 0.5 mL) was added and reaction wasstirred for 30 min. The reaction mixture was diluted with ether (15 mL)and Na₂SO₄ was added. The solution was filtered through Celite andsolvent removed in vacuo to provide the title compound 14 (191 mg, 95%)

Step 2: Synthesis of tert-butyl(S)-4-acetoxy-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(15): tert-butyl(S)-8-(chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(14, 171 mg, 0.483 mmol) was dissolved in CH₂Cl₂ (5.0 mL). Pyridine (213mg, 2.69 mmol) was added followed by acetyl chloride (114 mg, 1.45mmol). Reaction was stirred overnight and solution turned orange/brown.The solvent was removed in vacuo leaving a crude orange solid. The cruderesidue was purified by flash chromatography (0-100% EtOAc/heptane) toprovide after solvent removal provided desired product 15 (171.0 mg,89.4%)

Step 3: Synthesis of(S)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indol-4-ylacetate (16): tert-butyl(S)-4-acetoxy-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(15, 20 mg, 0.051 mmol) was taken up in 4N HCl in dioxane (1 mL). Thereaction was allowed to stand overnight and the solvent was removed toprovide crude target material 16 in quantitative yield and the materialwas used immediately

Preparation ofbicyclo[1.1.1]pentane-1,3-diylbis[carbonyl(8S)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6,4-diyl]Diacetate (17)

Step 1: Synthesis ofbicyclo[1.1.1]pentane-1,3-diylbis[carbonyl(8S)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6,4-diyl]diacetate (17): The title compound was prepared following generalprocedure B using 16, (17 mg, 0.051) and 6 (4.94 mg, 0.026 mmol, 0.0301mL, 1 M in THF), pyridine (14.3 0.181 mmol) and THE (1.0 mL), andpurification using medium pressure reverse phase C18 chromatography(Gradient: 0% to 80% acetonitrile in water with 0.02% TFA in each phase)providing desired product 17 (10.0 mg, 27%) LC-MS (Protocol B): m/z711.1 [M+H]⁺, retention time=1.12 minutes

Preparation of(R)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indol-4-ylAcetate (21)

Step 1: Synthesis of tert-butyl(R)-8-(chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(19): tert-Butyl(R)-4-(benzyloxy)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(18) [prepared as described in J. Am. Chem. Soc., 2007, 129,14092-14099.] (250 mg, 0.563 mmol) was taken up in THF (5.63 mL). 10%Pd/C (80 mg) was added. A freshly prepared 25% aqueous Formic acidammonium salt (500 mg, 2 mmol, 0.5 mL) was added and reaction wasstirred for 30 min. The reaction mixture was diluted with ether (15 mL)and Na₂SO₄ was added. The solution was filtered through Celite andsolvent removed in vacuo leaving a white solid of crude 19 (156 mg, 78%)

Step 2: Synthesis of tert-butyl(R)-4-acetoxy-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(20): tert-butyl(R)-8-(chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(19, 136 mg, 0.384 mmol) was dissolved in DCM (5.0 mL). Pyrdine (213 mg,2.69 mmol) was added followed by acetyl chloride (114 mg, 1.45 mmol).Reaction was stirred overnight and solution turned orange/brown. Thesolvent was removed in vacuo leaving a crude orange solid. The cruderesidue was purified by flash chromatography (0-100% EtOAc/heptane)provide after solvent removal provided desired product 20 (113.0 mg,74%)

Step 3: Synthesis of(R)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indol-4-ylacetate (21): tert-butyl(R)-4-acetoxy-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6-carboxylate(20, 25 mg, 0.051 mmol) was taken up in 4N HCl in dioxane (1 mL). Thereaction was allowed to stand overnight and the solvent was removed toprovide crude target material 21 in quantitative yield and the materialwas used immediately

Preparation ofBicyclo[1.1.1]pentane-1,3-diylbis[carbonyl(8R)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6,4-diyl]Diacetate (22)

Step 1: Synthesis ofBicyclo[1.1.1]pentane-1,3-diylbis[carbonyl(8R)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6,4-diyl]diacetate (22): The title compound was prepared following generalprocedure B using 21, (17 mg, 0.051 mmol) and 6 (4.94 mg, 0.026 mmol,0.0301 mL, 1 M in THF), pyridine (14.3 0.181 mmol) and THE (1.0 mL), andpurification using medium pressure reverse phase C18 chromatography(Gradient: 0% to 80% acetonitrile in water with 0.02% TFA in each phase)providing desired product 22 (8.6 mg, 19%) LC-MS (Protocol B): m/z 711.1[M+H]⁺, retention time=1.12 minutes

Preparation of(8S,8'S)-glutaroylbis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)Diacetate (24)

Step 1: Synthesis of(8S,8'S)-glutaroylbis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)Diacetate (24): A solution of 3 (10 mg, 0.025 mmol) was treated with 4MHCl (0.5 mL in dioxane) at rt for 1 h and concentrated in vacuo Theresidue was dissolved in THF (2 mL) and glutaryl chloride (2.13 mg,0.0125 mmol), was added followed by DIPEA (13 uL, 0.074 mmol). Theresulting mixture was stirred at rt for 30 min. The reaction mixture wasconcentrated and the residue was purified by silica gel chromatographyusing MeOH/DCM (0-10%) to give the product as off-white solid, which wastreated with MeOH, filtered to give product 24 as off-white solid 6 mg(70%). LC-MS (Protocol B): m/z 687.1 [M+H]⁺, retention time=1.11minutes.

¹H NMR (400 MHz, CDCl₃) δ=8.24 (s, 2H), 7.13 (s, 2H), 4.36 (d, J=10.5Hz, 2H), 4.17 (m, 2H), 4.06 (m, 2H), 3.74 (d, J=10.9 Hz, 2H), 3.39 (t,J=10.9 Hz, 2H), 2.80 (m, 2H), 2.66 (m, 2H), 2.55 (s, 6H), 2.37 (s, 6H),2.22 (m, 2H).

Preparation of(8R,8′R)-glutaroylbis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)Diacetate (25)

Step 1: Synthesis of(8R,8′R)-glutaroylbis(8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl)Diacetate (25):

A solution of 10 (10 mg, 0.025 mmol) was treated with 4M HCl (0.5 mL indioxane) at rt for 1 h and Concentrated in vacuo. The residue wasdissolved was dissolved in THF (2 mL), and glutaryl chloride (2.13 mg,0.0125 mmol) was added followed by Pyridine (12 mg, 0.15 mmol). Theresulting mixture was stirred at rt for 4 h., concentrated in vacuo andthe residue was purified by reverse phase HPLC (Method C) to give theproduct 25 as off-white solid 1.5 mg (17%). LC-MS (Protocol B): m/z687.1 [M+H]⁺, retention time=1.11

¹H NMR (400 MHz, CDCl₃)=8.24 (s, 2H), 7.13 (s, 2H), 4.37 (d, J=10.5 Hz,2H), 4.18 (m, 2H), 4.06 (m, 2H), 3.73 (d, J=10.9 Hz, 2H), 3.39 (t,J=10.9 Hz, 2H), 2.77 (m, 2H), 2.66 (m, 2H), 2.55 (s, 6H), 2.37 (s, 6H),2.22 (m, 2H).

Preparation of(8S,8'S)-glutaroylbis(8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6,4-diyl)Diacetate (26)

Step 1:(8S,8'S)-glutaroylbis(8-(chloromethyl)-2-methyl-7,8-dihydro-6H-thieno[2,3-e]indole-6,4-diyl)Diacetate (26): A solution of 15 (12 mg, 0.03 mmol) was treated with 4MHCl (0.5 mL in dioxane) at rt for 1 h. and Concentrated in vacuo. Theresidue was dissolved in THF (2 mL), and glutaryl chloride (2.5 mg,0.015 mmol) was added followed by DIPEA (16 uL, 0.09 mmol). Theresulting mixture was stirred at rt for 30 min, and concentrated invacuo and the residue was purified by ISCO using MeOH/DCM (0-10%) togive the product 26 as off-white solid 6 mg (60%): LC-MS (Protocol B):m/z 687.1 [M+H]⁺, retention time=1.11.

¹H NMR (400 MHz, CDCl₃) 3=8.12 (s, 2H), 6.84 (s, 2H), 4.32 (m, 2H), 4.2(m, 2H), 4.05 (d, J=10.9 Hz, 2H), 3.93 (m, 2H), 3.62 (m, 2H), 2.68(br.s., 4H), 2.56 (s, 6H), 2.37 (s, 6H), 2.19 (br.s., 2H).

Preparation of(8S)-8-(chloromethyl)-6-[(3-{[(8S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-nitrophenyl Carbonate (33)

Step 1: Synthesis of tert-butyl(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(2). To a stirring solution of tert-butyl(8S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate1 [prepared as described in J. Am. Chem. Soc., 2007, 129, 14092-14099.](575 mg, 1.30 mmol) in 18 mL of THF at 0° C., Palladium 10 wt. % oncarbon (250 mg) was added followed by slow dropwise addition of 2.0 mLof 25% ammonium formate in water. The reaction was allowed to stir at 0°C. for ˜90 minutes. Reaction was diluted with ether followed by theaddition of sodium sulfate. The reaction was filtered through a thin padof celite, which was then washed twice with ether. The organics wherecombined and then reduced down before being placed underneath vacuumyielding 2 (458 mg, quantitative) as off white solid. LC-MS (ProtocolA): m/z 352.2 [M−H], retention time=1.96 minutes.

Step 2: Synthesis of(8S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylacetate (4). To a stirring solution of 2 (209 mg, 0.591 mmol) in 8 mL ofdichloromethane at 0° C., acetyl chloride (0.0462 mL, 0.650 mmol) wasadded followed immediately by pyridine (0.0714 mL, 0.886 mmol). Thereaction was allowed to stir at 0° C. for ˜10 minutes. Reaction wasreduced down onto silica. Silica chromatography was then preformed(gradient: 0%-15% acetone in heptanes). Appropriate test tubes whereconcentrated and placed underneath high vacuum to produce a white solid.To this white solid 4M HCl in dioxane (10 mL, 40 mmol) was added and thereaction was allowed to stir at room temperature for ˜90 minutes.Reaction was diluted with heptane, concentrated in vacuo, and thenplaced underneath high vacuum to produce 4 (170 mg, 80% yield, 2 steps)as a light orange solid. LC-MS (Protocol A): m/z 296.1 [M+H]⁺, retentiontime=1.56 minutes. ¹H NMR (400 MHz, DMSO-d₆): δ 7.53 (s, 1H), 7.05 (s,1H), 4.27-4.19 (m, 1H), 3.93-3.86 (m, 1H), 3.80-3.75 (m, 1H), 3.72-3.64(m, 2H), 2.54 (s, 3H), 2.37 (s, 3H).

Step 3: Synthesis of methyl3-(chlorocarbonyl)bicyclo[1.1.1]pentane-1-carboxylate (28). Followinggeneral procedure A using3-(methoxycarbonyl)bicyclo[1.1.1]pentane-1-carboxylic acid 27 (86.8 mg,0.510 mmol), oxalyl chloride (0.0525 mL, 0.612 mmol), THF (6 mL), and 1drop of DMF, 28 was prepared as a white solid (97 mg, quant.). Crude 28was used as is without further purification.

Step 4: Synthesis of dibenzyl(8S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylphosphate (29). To a stirring solution of 2 (4.5 g, 13.4 mmol) in 8 mLof THF and 8 mL of acetonitrile, carbon tetrachloride (0.997 mL, 10.3mmol) was added followed by Hunig's base (0.512 mL, 2.94 mmol),dibenzylphosphite (0.974 mL, 4.41 mmol), and DMAP (18 mg, 0.147 mmol).The reaction was allowed to stir at room temperature for ˜10 minutes.Reaction was reduced down onto silica. Silica chromatography was thenpreformed (gradient: 0%-25% acetone in heptanes). Appropriate test tubeswhere concentrated and placed underneath high vacuum to produce a whitesolid. Crude material was dissolved in 5 mL of dichloromethane followedby the addition of trifluoroacetic acid (5 mL, 70 mmol). The reactionwas allowed to stir at room temperature for 60 seconds, immediatelyconcentrated in vacuo, and then placed underneath high vacuum producing29 (321 mg, 70% yield, 2 steps) as white and clear, oil and solid mix.LC-MS (Protocol A): m/z 514.1 [M+H]⁺, retention time=2.14 minutes.

Step 5: Synthesis of3-{[(8S)-4-{[bis(benzyloxy)phosphoryl]oxy}-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}bicyclo[1.1.1]pentane-1-carboxylicacid (30). Following general procedure B using 29 (315 mg, 0.502 mmol),28 (94.6 mg, 0.502 mmol), triethylamine (0.210 mL, 1.50 mmol) and THF(20 mL), and purification using silica gel chromatography (Gradient: 0%to 35% acetone in heptane). Appropriate test tubes where combined andconcentrated in vacuo producing a white solid. Material was dissolved inTHF (10 mL) followed by the addition of lithium hydroxide dissolved in2.5 mL of water. The reaction was allowed to stir at room temperaturefor ˜45 minutes. The reaction was diluted with dichloromethane andquenched through the addition of 1N HCl (aq.). Reaction was transferredto a separatory funnel. The organic layer was separated and the aqueouslayer was washed twice with dichloromethane. The organic layers wherecombined, washed once with brine, washed once with water, dried oversodium sulfate, filtered, concentrated in vacuo, and then placedunderneath high vacuum producing 30 (178 mg, 57% yield, 2 steps). LC-MS(Protocol A): m/z 652.2 [M+H]⁺, retention time=1.97 minutes.

Step 6: Synthesis of dibenzyl(8S)-6-{[3-(chlorocarbonyl)bicyclo[1.1.1]pent-1-yl]carbonyl}-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylphosphate (31). Following general procedure A using 30 (174 mg, 0.267mmol), oxalyl chloride (0.0298 mL, 0.347 mmol), THF (5 mL),dichloromethane (1 mL) and 1 drop of DMF, 31 was prepared as a whitesolid (182 mg, quant.). Crude 31 was used as is without furtherpurification.

Step 7: Synthesis of(8S)-6-[(3-{[(8S)-4-{[bis(benzyloxy)phosphoryl]oxy}-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylacetate (32). Following general procedure B using 31 (167 mg, 0.249mmol), 4 (99.3 mg, 0.299 mmol), triethylamine (0.104 mL, 0.747 mmol) andTHF (20 mL), and purification using silica gel chromatography (Gradient:0% to 50% acetone in heptane). Appropriate test tubes where combined andconcentrated in vacuo producing 32 (103 mg, 45%) a white solid. LC-MS(Protocol A): m/z 929.3 [M+H]⁺, retention time=2.44 minutes. ¹H NMR (400MHz, DMSO-d₆): δ 8.37 (s, 1H), 8.09 (s, 1H), 7.54-7.50 (m, 2H),7.39-7.33 (m, 10H), 5.23-5.14 (m, 4H), 4.53-4.46 (m, 2H), 4.38-4.25 (m,4H), 4.02-3.95 (m, 2H), 3.78-3.69 (m, 2H), 2.63 (s, 6H), 2.57-2.53 (m,6H), 2.39 (s, 3H).

Step 8: Synthesis of(8S)-8-(chloromethyl)-6-[(3-{[(8S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl4-nitrophenyl carbonate (33). To a stirring solution of 32 (99 mg, 0.11mmol) in 6 mL of methanol, 4M HCl in dioxane (6.0 mL, 20 mmol) wasadded. The reaction was allowed to stir at room temperature for ˜15minutes. Reaction was reduced and then placed underneath high vacuum. Toa stirring solution of crude material in 6 mL of dichloromethane and 6mL of THF at 0° C., p-nitrophenyl chloroformate (38.6 mg, 0.192 mmol)was added followed immediately by triethylamine (0.0742 mL, 0.532 mmol).The reaction was allowed to stir at 0° C. for ˜5 minutes and thenallowed to warm to room temperature while stirring. The reaction wasallowed to stir at room temperature for ˜10 minutes. Reaction wasreduced down. To a stirring solution of crude material in 3 mL ofdichloromethane, a solution of TFA (3.0 mL, 39 mmol) in 3 mL ofdichloromethane was added followed by the addition of thiophenol (0.109mL, 1.06 mmol). The reaction was allowed to stir at room temperature for˜6 hours. Reaction was reduced down. Crude material was diluted with afew milliliters of DMSO and then injected onto a 25 g C18 pre-column(which was previously equilibrated with acetonitrile and then water,with 0.02% TFA in each phase). Material was purified by medium pressurereverse phase C18 chromatography (Gradient: 20% to 65% acetonitrile inwater with 0.02% TFA in each phase) with the appropriate test tubesconcentrated using a genevac producing 33 (37 mg, 40%, 3 steps) as awhite solid. LC-MS (Protocol A): m/z 872.3 [M+H]⁺, retention time=1.86minutes. ¹H NMR (400 MHz, DMSO-d₆): δ 8.44 (s, 1H), 8.40-8.33 (m, 3H),7.78-7.72 (m, 2H), 7.59 (m, 1H), 7.47 (m, 1H), 4.53-4.44 (m, 2H),4.38-4.22 (m, 4H), 4.04-3.94 (m, 2H), 3.81-3.75 (m, 1H), 3.72-3.65 (m,1H), 2.62 (s, 6H), 2.58 (s, 3H), 2.54 (s, 3H).

Preparation ofN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N˜5˜-carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-ornithinamide (40)

Step 1: Synthesis ofN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl-N˜5˜-carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithinamide(36). To a round bottom flask containing1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oicacid, 34(628 mg, 1.45 mmol), 20 mL of dichloromethane, 2 mL of DMF, HATU(501 mg, 1.32 mmol) and Hunig's base (0.92 mL, 5.3 mmol) was added. Thereaction was allowed to stir at room temperature for 2 minutes beforethe addition ofL-valyl˜N˜5˜-carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithinamide, 35(500 mg, 1.32 mmol). The reaction was allowed to stir at roomtemperature for ˜90 minutes before being quenched through the additionof TFA. The reaction was concentrated to a smaller volume, diluted witha few mLs of DMSO and then injected onto a 25 g C18 pre-column (whichwas previously equilibrated with acetonitrile and then water, with 0.02%TFA in each phase). Material was purified by medium pressure reversephase C18 chromatography (Gradient: 5% to 40% acetonitrile in water with0.02% TFA in each phase) with the appropriate test tubes concentratedusing a genevac producing 36 (514 mg, 49%) as a clear solid. LC-MS(Protocol A): m/z 795.5 [M+H]⁺, retention time=1.01 minutes.

Step 2: Synthesis ofN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl˜N˜5˜-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(37). To a stirring solution of 36 (210 mg, 0.264 mmol) andbis(4-nitrophenyl) carbonate (161 mg, 0.528 mmol) in 4 mL of DMF,Hunig's base (0.096 mL, 0.554 mmol) was added. The reaction was allowedto stir at room temperature for ˜2 hours. The reaction was injected ontoa 25 g C18 pre-column (which was previously equilibrated withacetonitrile and then water, with 0.02% TFA in each phase). Material waspurified by medium pressure reverse phase C18 chromatography (Gradient:5% to 55% acetonitrile in water with 0.02% TFA in each phase) with theappropriate test tubes concentrated using a genevac producing 37(180 mg,71%) as a solid. LC-MS (Protocol A): m/z 960.5 [M+H]⁺, retentiontime=1.48 minutes.

Step 3: Synthesis ofN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl˜N˜5˜-carbamoyl-N-[4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl)phenyl]-L-ornithinamide(39). To a stirring solution of 37 (640 mg, 0.667 mmol) and 38 [preparedas described J. Med. Chem. 1992, 33, 559-567] (127 mg, 0.674 mmol) in 6mL of DMA, 2,6-Lutidine (0.154 mL, 1.33 mmol) was added followed byHunig's base (0.232 mL, 1.33 mmol) and HOAT (9.1 mg, 0.67 mmol). Thereaction was allowed to stir at room temperature for ˜15 minutes. Thereaction was injected onto a 25 g C18 pre-column (which was previouslyequilibrated with acetonitrile and then water, with 0.02% TFA in eachphase). Material was purified by medium pressure reverse phase C18chromatography (Gradient: 5% to 40% acetonitrile in water with 0.02% TFAin each phase) with the appropriate test tubes concentrated using agenevac producing 39 (564 mg, 84%) as a wax like white solid. LC-MS(Protocol A): m/z 1009.7 [M+H]⁺, retention time=1.43 minutes.

Step 4: Synthesis ofN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl˜N˜5˜-carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-ornithinamide(40). To a stirring mixture of 39 (470 mg, 0.466 mmol) in 6 mL ofdichloromethane, TFA (3.0 mL, 40 mmol) was added. The reaction wasallowed to stir at room temperature for ˜10 minutes. Reaction wasreduced down. Residue was purified by medium pressure reverse phase C18chromatography (Gradient: 5% to 30% acetonitrile in water with 0.02% TFAin each phase) with the appropriate test tubes concentrated using agenevac producing 40 (326 mg, 68%) as a white oil/solid mix. LC-MS(Protocol A): m/z 909.8 [M+H]⁺, retention time=0.91 minutes.

Preparation ofN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl˜N˜5˜-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(8S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide(41)

Step 1: Synthesis ofN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo-3,6,9,12,15,18-hexaoxahenicosan-21-yl]-L-valyl˜N˜5˜-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(8S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide(41). To a 2 dram vial containing 33 (8.0 mg, 0.0092 mmol) and 40 (10.3mg, 0.0101 mmol), 1.0 mL of DMA was added followed by Hunig's base(0.00639 mL, 0.0367 mmol), 2,6-Lutidine (0.00425 mL, 0.0367 mmol) andHOAT (1.25 mg, 0.0367 mmol). The reaction was allowed to stir at roomtemperature for ˜5 minutes. Crude reaction was injected onto a 4 g C18pre-column (which was previously equilibrated with acetonitrile and thenwater, with 0.02% TFA in each phase). Material was purified by mediumpressure reverse phase C18 chromatography (Gradient: 15% to 50%acetonitrile in water with 0.02% TFA in each phase) followed by a secondpurification by method A with the appropriate test tubes concentratedusing a genevac producing 41 (8.9 mg, 59%) as a white solid. LC-MS(Protocol A): m/z 1642.9 [M+2H]⁺, retention time=1.61 minutes.

Preparation of LP:(2S,3S,4S,5R,6S)-6-(((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-4-(((2-((((4-((23S,26S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (49)

Step 1: Synthesis of tert-butyl 5-chloro-5-oxopentanoate (44): To asolution of 5-(tert-butoxy)-5-oxopentanoic acid (110 mg, 0.58 mmol) inTHF (3 mL), was added oxalyl chloride (0.58 mL, 1.2 mmol, 2M in DCM) at0° C. followed by 1 drop of DMF. The mixture was stirred at 0° C. for 30min, and concentrated in vacuo to give the corresponding acid chloride44 as white solid.

Step 2. Synthesis of(2S,3R,4S,5S,6S)-2-(((S)-6-(tert-butoxycarbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (43)

To a solution of 2 (226 mg, 0.64 mmol) in DCM (23 mL), was added 4 A MS(1.17 g, powder, <5 micro, activated), and the mixture was stirred atroom temperature for 30 min. To the reaction mixture,alpha-D-glucuronide methyl ester 2,3,4-triacetate1-2,2,2-trichloroethanimidate (42, 367 mg, 0.77 mmol) was added, and themixture cooled to −25° C. And a solution of BF₃.Et₂O (0.13 mL, 0.32mmol) in DCM (10 mL) was added slowly and mixture was stirred at below−20° C. for 1 h. The reaction mixture was filtered and the solution wasconcentrated in vacuo. The residue was purified by silica gelchromatography using a gradient of 0-60% EtOAc in Heptanes to give theproduct 43 as a yellow solid 261 mg (61%).

LC-MS (Protocol B): m/z 692.1 (M+Na), retention time=1.09 min. ¹H NMR(400 MHz, CHLOROFORM-d) δ=7.29 (s, 1H), 7.13 (s, 1H), 5.40 (br. s., 3H),4.30 (d, J=7.4 Hz, 2H), 4.06-3.92 (m, 2H), 3.85-3.73 (m, 4H), 3.69 (d,J=10.5 Hz, 1H), 3.36 (t, J=10.5 Hz, 1H), 2.56 (s, 3H), 2.10 (s, 6H),2.08 (s, 3H), 1.62 (s, 9H).

Step 2. Synthesis of(2S,3R,4S,5S,6S)-2-(((S)-6-(5-(tert-butoxy)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (45)

A solution of 43 (261 mg, 0.39 mmol) was treated with 4M HCl in dioxane(3 mL) for 1 h. Concentrated in vacuo. and the residue was dissolved inTHF (3.0 mL) and a solution of acid chloride 44 (0.58 mmol) in THF (3.0mL) was added followed by TEA (0.163 mL, 1.2 mmol). The mixture wasstirred at 0° C. for 30 min. The mixture was concentrated, and theresidue was purified by silica gel chromatography using a gradient ofEtOAc (0-70%) in heptanes to give the product as off-white solid 224 mg(78%). LC-MS (Protocol B): m/z 740.2 [M+H]⁺, retention time=1.07minutes. ¹H NMR (400 MHz, CHLOROFORM-d) δ=8.26 (br. s., 1H), 7.13 (s,1H), 5.45-5.25 (m, 4H), 4.32 (d, J=9.0 Hz, 2H), 4.12 (t, J=8.8 Hz, 1H),4.05 (d, J=8.6 Hz, 1H), 3.79-3.66 (m, 5H), 3.33 (t, J=10.9 Hz, 1H), 2.63(d, J=7.0 Hz, 1H), 2.58-2.47 (m, 4H), 2.45-2.35 (m, 2H), 2.06 (m 11H),1.47 (s, 9H).

Step 3. Synthesis of(2S,3R,4S,5S,6S)-2-(((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triylTriacetate (46)

A solution of 45 (116 mg, 0.16 mmol) was treated with DCM (1.5 mL) andTFA (1 mL) at rt for 1 h, concentrated in vacuo and the residue wasdissolved in THF at 0° C., and oxalyl chloride (0.16 mL, 0.32 mmol, 2Min DCM), and DMF (1 drop) were added. The mixture was stirred at 0° C.for 30 min and concentrated in vacuo to give the corresponding acidchloride.

In a separate vial compound 2 (83 mg, 0.24 mmol) was treated with 4M HCl(2 mL) in dioxane at rt for 1 h and concentrated in vacuo was and theresidue was dissolved in THF (5 mL) at 0° C., and Et3N (100 uL, 0.78mmol) was added, followed by a solution of the above acid chloride inTHF (5 mL). The mixture was stirred at 0° C. for 20 min. The mixture wasconcentrated, and the residue was purified by silica gel chromatographyusing a gradient of EtOAc (0-100%) in heptanes to give the product 46 asoff-white solid 114 mg (79%). LC-MS (Protocol B): m/z 919.1 [M+H]⁺,retention time=1.05 minutes, ¹H NMR (400 MHz, DMSO-d6) δ=10.41 (s, 1H),8.22 (s, 1H), 7.93 (s, 1H), 7.47 (s, 1H), 7.38 (s, 1H), 5.81-5.70 (m,4H), 5.63-5.52 (m, 1H), 5.19 (t, J=8.6 Hz, 1H), 5.12 (t, J=9.8 Hz, 1H),4.76 (d, J=9.8 Hz, 1H), 4.30-4.16 (m, 3H), 3.86 (m, 2H), 3.68 (s, 3H),3.64-3.51 (m, 2H), 2.73 (br. s., 1H), 2.62 (m, 2H), 2.50 (s, 6H),2.10-2.02 (m, 12H).

Step 4. Synthesis of(2S,3R,4S,5S,6S)-2-(((S)-6-(5-((S)-4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (47): To a solution of 46 (50 mg, 0.054 mmol) in THF (3 mL)at 0° C., was added a solution of 4-nitrophenyl chloroformate (22 mg,0.10 mmol) in DCM (0.5 mL), followed by DIPEA (57 uL, 0.33 mmol), andthe mixture was stirred at 0° C. for 1 h. To the above mixture was addeda solution of 38 [prepared as described J. Med. Chem. 1992, 33, 559-567](31 mg, 0.16 mmol) in THF (0.5 mL), and the mixture was stirred at 0° C.for 30 min and concentrated in vacuo, and the residue was purified waspurified by silica gel chromatography using a gradient of EtOAc (0-100%)in heptanes to give the product 47 as white solid 56 mg (91%). LC-MS(Protocol B): m/z 1150.2 [M+NH₄], retention time=1.14 minutes

Step 5: Synthesis of(2S,3S,4S,5R,6S)-6-(((S)-6-(5-((S)-4-(((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (48): To a solution of 47 (56 mg, 0.049 mmol) in THF/MeOH (1/1, 6mL) at 0° C., was added a solution of LiOH.H2O (21 mg, 0.49 mmol) inwater (0.5 mL). The mixture was stirred at 0° C. for 1 h. Acetic acid(50 mg) was added and the reaction concentrated in vacuo to give crudeproduct 48 as white solid 45 mg (90%). LC-MS (Protocol B): m/z 1014.9[M+Na], retention time=1.0 minutes

Step 6: Synthesis of(2S,3S,4S,5R,6S)-6-(((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-4-(((2-((((4-((23S,26S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (49) and(8S)-8-(chloromethyl)-6-{5-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-5-oxopentanoyl}-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylbeta-D-glucopyranosiduronic acid (57): Compound 48 (23 mg, 0.02 mmol)was treated with pre-cooled TFA (1 mL) at 0° C. for 5 min. andconcentrated in vacuo. The residue was dissolved in DMF (2 mL), andcompound 37(19 mg, 0.02 mmol), lutidine (14 uL, 0.12 mmol), DIPEA (21uL, 0.12 mmol) and HOAt (2.7 mg, 0.02 mmol) were added and. the mixturewas stirred at 30° C. for 1 h The mixture was purified by reverse phaseHPLC (Method C) to give compound 49 as off-white solid 8 mg (20%). LC-MS(Protocol B): m/z 1714.6 [M+H]⁺, retention time=0.89 minutes andcompound 57 as a gum 7 mg (50%): LC-MS (Protocol B): m/z 779.1 [M+H]⁺,retention time=0.90 minutes

Preparation of:(S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl(4-((23S,26S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzyl)ethane-1,2-diylbis(methylcarbamate) (56)

Step 1: Synthesis of methyl 5-chloro-5-oxopentanoate (51):5-methoxy-5-oxopentanoic acid (128 mg, 0.88 mmol) was dissolved in THF(5 mL) at 0 C, and oxalyl chloride (0.9 mL, 1.8 mmol, 2M in DCM) and DMF(1 drop) were added and the mixture was stirred at 0° C. for 30 min.Concentrated in vacuo to give the correspond acid chloride 51 as whitesolid which was used without further purification.

Step 2. Synthesis of tert-butyl(S)-4-((bis(benzyloxy)phosphoryl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(50)

To a solution of 2 (260 mg, 0.74 mmol) in ACN (8 mL) and THF (8 mL), wasadded CCl₄ (1 mL), DIPEA (0.52 mL, 2.94 mmol), dibenylphosphite (1.03mL, 4.41 mmol), and DMAP (18 mg). The mixture was stirred at rt for 15min, concentrated in vacuo the residue was purified by silica gelchromatography using a gradient (0-60%) of EtOAc in heptanes to give theproduct 50 as colorless oil 365 mg (81%). LC-MS (Protocol B): m/z 631.2[M+H]⁺, retention time=1.18 minutes

Step 3. Synthesis of methyl(S)-5-(4-((bis(benzyloxy)phosphoryl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoate(52)

To a solution of 50 (365 mg, 0.58 mmol) in DCM (3 mL) was added TFA (3mL) and the mixture was stirred at rt for 2 min and concentrated invacuo. The residue was dissolved in THF (5.0 mL) and a solution of acidchloride 51 (0.88 mmol) in THF (5.0 mL) followed by Et₃N (0.37 mL, 0.24mmol) was added and the mixture was stirred at 0° C. for 30 min. Thereaction was concentrated and the residue was purified by silica gelchromatography using a gradient (0-80%) of EtOAc in heptanes to give theproduct 52 as colorless oil 240 mg (64%). LC-MS (Protocol B): m/z 642.1[M+H]⁺, retention time=1.09 minutes

Step 4. Synthesis of(S)-5-(4-((bis(benzyloxy)phosphoryl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoicacid (53)

To a solution of 52 (235 mg, 0.37 mmol) in THF (10 mL) at 0° C., added asolution of LiOH/H₂O (155 mg, 3.7 mmol) in water (2.5 mL), and themixture was stirred at 0° C. for 1.5 h. The mixture was diluted withDCM, acidified by 1M HCl, and organic layer was separated, and theaqueous phase was extracted with DCM tow times. Combined organic phaseswere dried over MgSO4. The mixture was concentrated in vacuo and theresidue which was purified by reverse phase HPLC (Method C) to give theproduct 53 as off-white foam 27 mg (12%). LC-MS (Protocol B): m/z 628.1[M+H]⁺, retention time=1.01 minutes

Step 5. Synthesis of dibenzyl((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)phosphate (54)

To a solution of 53 (27 mg, 0.043 mmol) in THF (5 mL), at 0° C., wasadded oxalyl chloride (0.043 mL, 0.086 mmol, 2M in DCM) and DMF (1drop). The mixture was stirred at 0° C. for 30 min, and concentrated invacuo to give the corresponding acid chloride as yellow foam.

In a separate vial, compound 2 (23 mg, 0.064 mmol) was treated with 4MHCl (1 mL) at rt for 1 h, and concentrated in vacuo. The residue wasdissolved in THF (5 mL), cooled to 0° C., and added to a solution of theabove acid chloride in THF (5 mL) and Et₃N (0.018 mL, 0.13 mmol). Themixture was concentrated in vacuo, and the residue was purified byreverse phase HPLC (Method C) to give the product 54 as an off-whitesolid 28 mg (75%). LC-MS (Protocol B): m/z 863.2 [M+H]⁺, retentiontime=1.16 minutes

Step 6. Synthesis of(S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl(4-nitrophenyl) carbonate (55) and(8S)-8-(chloromethyl)-6-{5-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-5-oxopentanoyl}-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yldihydrogen phosphate (61)

To a solution of 54 (40 mg, 0.046 mmol) in THF (5 mL), at 0° C., wasadded a solution of 4-nitrophenyl chloroformate (19.4 mg, 0.092 mmol) inDCM (0.5 mL), and DIPEA (0.049 mL, 0.28 mmol). The mixture was stirredat 0° C. for 30 min. The reaction was concentrated in vacuo and theresidue was purified by silica gel chromatography using a gradient(0-60%) of acetone in heptanesto give intermediate PNP carbonate aswhite solid 48 mg. It was dissolved in DCM (2 mL), and TFA (2 mL) andthiophenol (0.047 mL, 0.46 mmol) were added and the mixture was stirredat rt for 3 h. The reaction was concentrated in vacuo, and the residuewas purified by reverse phase HPLC (Method C) to give 17.8 mg (46%) ofthe product 55 as white solid (17.8 mg, 46%). LC-MS (Protocol B): m/z848.2 [M+H]⁺, retention time=1.09 minutes and 5.2 mg (17%) of product 61as a gum LC-MS (Protocol B): m/z 683.2 [M+H]⁺, retention time=0.93minutes

Step 7. Synthesis of(S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methy-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl(4-((23S,26S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzyl)ethane-1,2-diylbis(methylcarbamate) (56)

To a solution of 55 (10.5 mg, 0.012 mmol) in DMF (1 mL), was added 40(13.9 mg, 0.014 mmol), lutidine (0.009 mL, 0.074 mmol), DIPEA (0.013 mL,0.074 mmol) and HOAt (1.7 mg, 0.012 mmol). The mixture was stirred at rtfor 20 min, concentrated in vacuo, and the residue was purified byreverse phase HPLC (Method C) to give the product 56 as white foam 12 mg(60%). LC-MS (Protocol B): m/z 1619.6 [M+2H]⁺, retention time=0.95minutes

Preparation ofpentacyclo[4.2.0.0^(˜)2,5^(˜).0^(˜)3,8^(˜).0^(˜)4,7^(˜)]octane-1,4-diylbis[carbony(8S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl]Diacetate (60)

Step 1: Synthesis ofpentacyclo[4.2.0.0^(˜)2,5^(˜).0^(˜)3,8^(˜).0^(˜)4,7^(˜)]octane-1,4-diylbis[carbonyl(8S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6,4-diyl]diacetate(60): A solution of 3 (47 mg, 0.12 mmol) was treated with 4M HCl (1.5 mLin dioxane) at rt for 90 min and concentrated in vacuo and the residuewas dissolved in DCM (4 mL) and TEA (0.025 mL) and added to a solutionof 1,4-Cubanedicarboxcylic acid (11.5 mg, 0.06 mmol, 72) was dissolvedin 1 mL of anhydrous dichloromethane followed by addition of HATU (47mg, 0.12 mmol). The reaction was stirred at room temperature for 16 h.The mixture was purified by reverse phase HPLC Column: (PhenomenexColumn Luna C18 5 u 150×21.5 mm, Gradient 20%-90% AcCN/Water (with 0.02%AcOH in each) over 20 min (plus an initial 6 minute isocratic time at20% AcCN/water) with 0.02% to afford 3.1 mg 60 (white solid, 7%). LC-MS(Protocol A): m/z: 747.1 (M+H)+, retention time=2.39 min

Preparation of(S)-8-(chloromethyl)-6-(5-((S)-1-(chloromethyl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)-1,2-dihydro-3H-benzo[e]indol-3-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ylDihydrogen Phosphate (62)

Step 1: Synthesis of(S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole(63): To a stirred solution of compound 1 (300 mg, 0.676 mmol) in dryDCM (5 mL) was added dropwise 4.0 M HCl in EtOAc (5 mL) at 0° C. Afteraddition, the mixture was stirred at r.t for 1.5 h. The mixture wasconcentrated in vacuum then co-evaporated with DCM once to give product63 (260 mg, 100%) and was used as such in next step.

Step 2: Synthesis of tert-butyl(S)-5-(4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoate(63)

In a round bottom flask containing tert-butyl(S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxylate(1) (500 mg, 1.13 mmol) was added 25% TFA in DCM (10 mL). The reactionwas stirred for 30 min. The reaction was concentrated and placed onunder vacuum for 30 min to give the de-boc residue 63 which was taken upin 5 mL of DCM and used in the step below.

In a round bottom flask purged with N₂, containing5-(tert-butoxy)-5-oxopentanoic acid (212 mg, 1.13 mmol)) in 5 mL ofanhydrous DCM was added oxalyl chloride (0.101 mL, 1.13 mmol). To thissolution was added 1 drop of DMF and the system was stirred for 3 hours.Noticed gas formation immediately. The reaction was concentrated byvacuum to give crude acid chloride 44. which was taken up in DCM andadded to a round bottom flask containing the above described deprotectedresidue 63 and TEA (0.144 mL). The reaction was stirred at roomtemperature for 2 hours. The crude reaction mixture was concentrated byvacuum and taken up in 25 mL of DCM and transferred to a separatoryfunnel. Washed organic layer with 1M HCl (3×), water (3×), and brine(2×). Dried organic layer over sodium sulfate, filtered and concentratedthe filtrate to a crude solid. The crude products was purified by silicagel chromatography (Gradient: 0% to 10% MeOH in DCM) to give 64 as ayellow solid (525 mg, 90%). LC-MS (Protocol A): m/z 514 [M+H]⁺,retention time=2.43 minutes.

Step 3: Synthesis of tert-butyl(S)-5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate(66).

In a round bottom flask tert-butyl(S)-5-amino-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate(1000 mg, 3.0 mmol, 65) (prepared as described in WO 2015023355) in 15mL of DCM was added (9H-fluoren-9-yl)methyl(S)-(1-chloro-1-oxopropan-2-yl)carbamate (991 mg, 3.0 mmol followed by1.2 mL of Hunigs base. The reaction was stirred for 1 hour andconcentrated to a crude glass. The crude reaction mixture was purifiedby silica gel chromatography (gradient: 0% to 10% MeOH in DCM) to give66 as a white solid (1529 mg, 80%). LC-MS (Protocol A): m/z 626 [M+H]⁺,retention time=2.32 minutes.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl((S)-1-(((S)-3-(5-((S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)amino)-1-oxopropan-2-yl)carbamate(67).

To a stirring solution of tert-butyl(S)-5-(4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoate(500 mg, 0.973 mmol, 64) in 10 mL of DCM was added 2.5 mL of TFA and thereaction was stirred for 3 hours. Upon completion the reaction wasconcentrated under vacuum to a pale white solid. The solid was thentaken up in 5 mL of anhydrous DCM and oxalyl chloride (0.32 mL, 0.93mmol). The reaction was stirred for 3 hours and concentrated undervacuum to a white solid. tert-butyl(S)-5-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-1-(chloromethyl)-1,2-dihydro-3H-benzo[e]indole-3-carboxylate(0.973 mmol, 66) was taken up in 25% TFA in DCM (5 mL) and stirred for30 min. The reaction was concentrated under vacuum and taken back up in5 mL of DCM. Hunigs base (0.32 mL) was added followed by the acidchloride previously described. The reaction was stirred for 2 hours.Upon completion the reaction mixture was concentrated under vacuum. Thecrude products was purified by silica gel chromatography (Gradient: 0%to 10% MeOH in DCM) to give 67 as a white solid (510 mg, 54%). %). LC-MS(Protocol A): m/z 965 [M+H]⁺, retention time=2.61 minutes.

Step 5: Synthesis of tert-butyl((S)-1-(((S)-1-(((S)-3-(5-((S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(68).

To a stirring solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-3-(5-((S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)amino)-1-oxopropan-2-yl)carbamate(500 mg, 0.518 mmol, 67) in 5 mL of DCM was added 5 mL of Diethylamine.The reaction was stirred for 3 hours and concentrated under vacuum to ayellow solid. The yellow crude solid was taken up in 10 mL of anyhdrousTHF followed by 2,5-dioxopyrrolidin-1-yl(tert-butoxycarbonyl)-L-valinate (163 mg, 0.518 mmol) followed by TEA(0.2 mL). The reaction was stirred at 70 degrees Celsius for 4 hours.Upon completion the reaction mixture was concentrated under vacuum. Thecrude products was purified by silica gel chromatography (Gradient: 0%to 10% MeOH in DCM) to give 68 as a white solid (198 mg, 40%). %). LC-MS(Protocol A): m/z 942 [M+H]⁺, retention time=2.49 minutes.

Step 6: Synthesis of tert-butyl((S)-1-(((S)-1-(((S)-1-(chloromethyl)-3-(5-((S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(69).

A stirring solution of tert-butyl((S)-1-(((S)-1-(((S)-3-(5-((S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(325 mg, 0.345 mmol, 68) in 7 mL of THF under nitrogen was cooled to 0 Cusing an ice bath. Palladium 10 wt. % on activated carbon (10 mg) wasthen added followed by the slow drop wise addition of 0.5 mL of 25%ammonium formate in water. The reaction was allowed to stir at 0 C. for1 hour. Upon completion the reaction mixture was filtered through a padof celite and the filtrate concentrated under vacuum. The crude productswas purified by silica gel chromatography (gradient: 0% to 10% MeOH inDCM) to give 69 as a yellow solid (181 mg, 61%). LC-MS (Protocol A): m/z852 [M+H]⁺, retention time=2.18 minutes.

Step 7: Synthesis of tert-butyl((S)-1-(((S)-1-(((S)-3-(5-((S)-4-((bis(benzyloxy)phosphoryl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(70).

To a stirring solution of tert-butyl((S)-1-(((S)-1-(((S)-1-(chloromethyl)-3-(5-((S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(165 mg, 0.193 mmol, 69) in 10 mL of THF and 10 mL of AcCN, carbontetrachloride (2.04 mL, 21.0 mmol) was added followed by Huenig's base(1.12 mL, 6.45 mmol), dibenzylphosphite (320 mg, 1.16 mmol) and DMAP(catalytic). The reaction was allowed to stir at room temperature for 20minutes. The reaction was concentrated to a crude glass. The crudereaction mixture was purified by silica gel chromatography (gradient: 0%to 10% MeOH in DCM) to give 70 as a white glass (51 mg, 24%). LC-MS(Protocol A): m/z 1113 [M−H]⁻, retention time=2.50 minutes.

Step 8: Synthesis of(S)-8-(chloromethyl)-6-(5-((S)-1-(chloromethyl)-5-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)-1,2-dihydro-3H-benzo[e]indol-3-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yldihydrogen phosphate (62).

In a round bottom flask equipped with a stir bar, tert-butyl((S)-1-(((S)-1-(((S)-3-(5-((S)-4-((bis(benzyloxy)phosphoryl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(40 mg 0.036 mmol, 70) was taken up in 5 mL of DCM. TFA (2.5 mL) and 2drops of thiophenol were added and the reaction was stirred for 6 hours.The crude material was concentrated under vacuum. The crude residue wastaken up in 3 mL of DMF and pentafluorophenyl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (71) (13.6 mg, 0.036mmol) was added followed by TEA (0.036 mmol). The reaction was stirredfor 1 hour. The reaction was concentrated under vacuum and purified byHPLC Method to give 62 as a white solid (25 mg, 68%), retentiontime=7.115 minutes. LC-MS (Protocol A): m/z 1025 [M+H]⁺, retentiontime=2.01 minutes

Preparation ofN˜2˜-acetyl-N˜6˜-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysyl-L-valyl-N—5˜-carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-ornithinamide(74)

Step 1: Synthesis ofN˜2˜-acetyl-N˜6˜-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysyl-L-valyl-N˜5˜-carbamoyl-N-[4-(4,7,10,10-tetramethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl)phenyl]-L-ornithinamide(72A): To a stirred solution ofN˜2˜-acetyl-N˜6˜-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysyl-L-valyl˜N˜5˜-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(72) (Prepared as described in U.S. Pat. No. 9,169,264) (1.00 g, 1.07mmol) in DMF (20 mL) was added 38 [prepared as described J. Med. Chem.1992, 33, 559-567] (241 mg, 1.28 mmol) at 0° C. under N₂ balloon. Theresulting mixture was stirred at 0° C. for 30 mins. The mixture waspoured into TBME (200 mL). The resulting white suspension was filteredand washed with TBME (200 mL) to afford the title compound 72A (750 mg,71.3%) as a yellow solid

Step 2: Synthesis ofN˜2˜-acetyl-N˜6˜-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysyl-L-valyl-N˜5˜-carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-ornithinamide(74): To a stirred suspension of compound 72A (10.00 g, 10.1 mmol) inDCM(60.0 mL) was added TFA (50.0 mL) at 0° C. The resulting solution wasstirred at 0° C. for 40 min. The resulting suspension was filtered. Thefilter cake was washed with TBME (200 mL) and dried in vacuum to drynessto afford crude product The crude product was purified bypreparative-HPLC (Column: Phenomenex Synergi Max-RP 250×50 mm, 10 um,Gradient: 25% to 55% acetonitrile in water with 0.1% TFA in each phaseover 17.5 min and hold for 8 min at 100% acetonitrile in watercontaining 0.1% TFA, Flow rate: 80 mL/min) to give the title compound 74(5.2 g, 51.3% yield) as a white solid ¹H NMR (400 MHz, DMSO-d6) □ 10.03(s, 1H), 8.47 (br. s., 2H), 8.10 (d, J=7.0 Hz, 1H), 8.03 (d, J=7.8 Hz,1H), 7.89 (d, J=7.5 Hz, 2H), 7.69 (d, J=7.3 Hz, 3H), 7.60 (d, J=8.5 Hz,2H), 7.45-7.38 (m, 2H), 7.36-7.29 (m, 4H), 7.26 (t, J=5.4 Hz, 1H), 6.02(br. s., 1H), 5.01 (s, 2H), 4.43-4.33 (m, 1H), 4.32-4.16 (m, 5H), 3.49(t, J=6.0 Hz, 2H), 3.06-2.92 (m., 6H) 2.86 (s, 3H), 2.62-2.53 (m, 3H),1.99 (dd, J=6.5, 13.3 Hz, 1H), 1.85 (s, 3H), 1.76-1.27 (m, 10H), 0.83(d, J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H)

Preparation of LP:(2S,3S,4S,5R,6S)-6-(((S)-6-(5-((S)-4-(((2-((((4-((S)-2-((S)-2-((S)-2-acetamido-6-aminohexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (76)

Step 1: Synthesis of(2S,3R,4S,5S,6S)-2-(((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(((4-nitrophenoxy)carbonyl)oxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (73)

To a solution of 46 (120 mg, 0.13 mmol) in THF (5 mL) at 0° C., wasadded a solution of 4-nitrophenyl chloroformate (55 mg, 0.26 mmol) inDCM (0.5 mL), followed by Et₃N (109 uL, 0.78 mmol), and the mixture wasstirred at 0° C. for 30 min. The mixture was concentrated in vacuum, andthe residue was purified by reverse phase HPLC (Method C) to give theproduct 73 as white solid 109 mg (77%). LC-MS (Protocol B): m/z 1086.5(m+H), retention time=1.24 min

Step 2: Synthesis of(2S,3R,4S,5S,6S)-2-(((S)-6-(5-((S)-4-(((2-((((4-((9S,12S,15S)-9-acetamido-1-(9H-fluoren-9-yl)-12-isopropyl-3,10,13-trioxo-15-(3-ureidopropyl)-2-oxa-4,11,14-triazahexadecan-16-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (75)

Compound 73 (50 mg, 0.046 mmol) was dissolved in DMF (2 mL), and to itwas added4-((9S,12S,15S)-9-acetamido-1-(9H-fluoren-9-yl)-12-isopropyl-3,10,13-trioxo-15-(3-ureidopropyl)-2-oxa-4,11,14-triazahexadecan-16-amido)benzylmethyl(2-(methylamino)ethyl)carbamate (74, 55 mg, 0.055 mmol), lutidine(0.021 mL, 0.18 mmol), DIPEA (0.032 mL, 0.18 mmol) and HOAt (6 mg, 0.046mmol). The mixture was stirred at rt for 1 h. The mixture wasconcentrated in vacuum, and the residue was purified by reverse phaseHPLC (Method C) to give the product 75 as white powder after freeze dry72 mg (85%). LC-MS (Protocol B): m/z 1833.3 (M+H)retention time=1.17 min¹H NMR (400 MHz, DMSO-d6) 5=10.02 (br. s., 1H), 8.22 (s, 1H), 8.19-8.13(m, 1H), 8.10 (d, J=6.6 Hz, 1H), 8.02 (d, J=7.8 Hz, 1H), 7.89 (d, J=7.4Hz, 2H), 7.69 (d, J=7.0 Hz, 3H), 7.57 (d, J=7.0 Hz, 2H), 7.54-7.38 (m,5H), 7.37-7.30 (m, 3H), 7.26 (br. s., 3H), 5.99 (br. s., 1H), 5.74 (d,J=7.0 Hz, 1H), 5.64-5.49 (m, 1H), 5.20 (t, J=8.8 Hz, 1H), 5.13 (t, J=9.6Hz, 1H), 5.04 (br. s., 1H), 5.00 (br. s., 1H), 4.81-4.70 (m, 1H), 4.39(br.s., 2H), 4.32-4.11 (m, 14H), 3.95-3.80 (m, 3H), 3.62 (br. s., 1H),3.56 (br. s., 1H), 3.48 (br. s., 2H), 3.12 (br. s., 1H), 3.03 (br. s.,2H), 3.00-2.91 (m, 6H), 2.88 (br. s., 3H), 2.82-2.67 (m, 4H), 2.67-2.55(m, 7H), 2.10 (s, 1H), 2.08-1.91 (m, 14H), 1.85 (s, 3H), 1.79-1.64 (m,2H), 1.61 (br. s., 2H), 1.54-1.32 (m, 5H), 1.31-1.23 (m, 2H), 1.23-1.12(m, 2H), 0.84 (d, J=6.6 Hz, 3H), 0.87 (d, J=6.6 Hz, 3H).

Step 3. Preparation of LP:(2S,3S,4S,5R,6S)-6-(((S)-6-(5-((S)-4-(((2-((((4-((S)-2-((S)-2-((S)-2-acetamido-6-aminohexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (76)

Compound 75 (44 mg, 0.024 mmol) was dissolved in DMF (1 mL) and THF (5mL) and MeOH (1 mL), cooled to 0° C., LiOH.H₂O (10 mg, 0.24 mmol) wasadded and the mixture was stirred at 0° C. for 60 min. The mixture wasacidified by adding HOAc (30 uL), and concentrated in vacuum. Theresidue was purified by reverse phase HPLC (Method C) to give 25.0 mg(66%) of the product 76 as white powder.

LC-MS (Protocol B): m/z 1471.1 (M+2H)⁺ retention time=0.75 min ¹H NMR(500 MHz, DMSO-d6) δ=8.09 (br. s., 2H), 7.97 (d, J=8.1 Hz, 1H), 7.68(br. s., 2H), 7.48 (d, J=7.6 Hz, 1H), 7.44-7.39 (m, 1H), 7.39-7.31 (m,2H), 7.22-7.12 (m, 1H), 5.97 (br. s., 1H), 5.45-5.32 (m, 2H), 5.18 (br.s., 1H), 5.06 (d, J=5.6 Hz, 1H), 4.98 (br. s., 1H), 4.92 (br. s., 1H),4.31 (br. s., 1H), 4.26-4.05 (m, 8H), 3.82 (d, J=10.0 Hz, 1H), 3.76 (d,J=10.5 Hz, 1H), 3.70 (d, J=7.6 Hz, 1H), 3.66-3.57 (m, 1H), 3.57-3.44 (m,5H), 3.43-3.36 (m, 2H), 3.36-3.28 (m, 3H), 3.12-3.07 (m, 2H), 3.05 (br.s., 1H), 3.00-2.91 (m, 2H), 2.88 (d, J=9.8 Hz, 3H), 2.80 (s, 2H),2.72-2.59 (m, 4H), 2.59-2.37 (m, 22H), 2.02 (s, 2H), 1.97-1.83 (m, 3H),1.78 (s, 3H), 1.69 (td, J=3.2, 6.4 Hz, 2H), 1.66-1.48 (m, 3H), 1.47-1.32(m, 4H), 1.32-1.14 (m, 4H), 0.76 (d, J=6.4 Hz, 3H), 0.79 (d, J=5.9 Hz,3H).

Preparation of4-((S)-2-((S)-2-((S)-2-acetamido-6-aminohexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)ethane-1,2-diylbis(methylcarbamate) (77)

Step 1. Synthesis of(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((S)-6-(tert-butoxycarbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)tetrahydro-2H-pyran-3,4,5-triylTriacetate (78)

Compound 2 (300) mg, 0.85 mmol) was dissolved in DCM (30 mL), and 4A MS(1.5 g, powered, <5 micro, activated) was added and the mixture wasstirred at room temperature for 30 min. To the reaction mixture,alpha-D-galactopanose, 2,3,4,6-tetraacetate1-2,2,2-trichloroethanimidate (449 mg, 93%, 0.85 mmol) was added, andmixture was cooled to −15° C. and a solution of BF₃.Et₂O (0.052 mL, 0.42mmol) in DCM (5 mL) was added slowly, and the reaction mixture wasstirred at −15° C.-−20° C. for 1 h. The reaction mixture was filteredoff through a pad of Celite, washed with Acetone, concentrated to give aresidue. The residue was treated with MeOH, and concentrated in vacuo togive 580 mg (100%) of product 78 as an off-white solid

LC-MS (Protocol B): m/z: 706.4 (M+Na) retention time=1.09 min

Step 2. Synthesis of(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (79)

Compound 78 (580 mg, 0.85 mmol) was treated with a solution of 4M HCl indioxane (5 mL) for 30 min and mixture was concentrated in vacuum to givethe crude product 79 as green solid which was used without furtherpurification

LC-MS (Protocol B): m/z 584.3 (M+H), retention time=0.94 min. 1H NMR(400 MHz, METHANOL-d4) δ=7.53 (s, 1H), 7.22 (s, 1H), 5.57-5.46 (m, 3H),5.34 (dd, J=2.0, 9.0 Hz, 1H), 4.53 (d, J=6.6 Hz, 2H), 4.42 (t, J=6.4 Hz,1H), 4.25 (dt, J=6.4, 11.2 Hz, 2H), 4.18-4.01 (m, 5H), 3.74 (dd, J=8.6,11.7 Hz, 2H), 3.69 (s, 14H), 2.68-2.58 (m, 4H), 2.23 (s, 3H), 2.19-2.14(m, 2H), 2.14-2.07 (m, 7H), 2.07-1.94 (m, 6H), 1.33 (s, 3H).

Step 3. Synthesis of(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((S)-6-(5-(tert-butoxy)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)tetrahydro-2H-pyran-3,4,5-triylTriacetate (80)

A solution of 5-(tert-butoxy)-5-oxopentanoic acid (44, 191 mg, 1.02mmol) in THF (10 mL) was cooled to 0° C. and oxalyl chloride (1.02 mL,2M in DCM) was added followed by 1 drop of DMF. The mixture was stirredat 0° C. for 30 min, and concentrated in vacuum to give thecorresponding acid chloride as wax. This was dissolved in THF (10 mL),and added to a solution of 79 (526 mg, 0.85 mmol) in THF (10 mL), andfollowed by Et₃N (0.354 mL, 2.54 mmol). The mixture was stirred at 0° C.for 30 min. The mixture was concentrated, and the residue was purifiedby silica gel chromatography using a gradient of 0% to 70% Ethyl Acetatein Heptanes to give the 448 mg (70%) product 80 as off-white solid

LC-MS (Protocol B): m/z 754.4 (M+H), retention time=1.06 min

Step 4. Synthesis of(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)tetrahydro-2H-pyran-3,4,5-triylTriacetate (84)

A solution of compound 80 (448 mg, 0.59 mmol) was treated with DCM (3mL) and TFA (2 mL) at rt for 1 h and the mixture was concentrated invacuum to give the free acid 81 as green solid 212 mg. Acid 81 (167 mg,0.24 mmol) was dissolved in THF (5 mL) and the solution cooled to 0° C.and oxalyl chloride (0.24 mL, 2M in DCM), followed by DMF (1 drop) wereadded. The mixture was stirred at 0° C. for 30 min, and concentrated invacuum to give the corresponding acid chloride 82 which was used withoutfurther purification in next step

A solution of compound 2 (102 mg, 0.28 mmol) in 4M HCl (2 mL) in dioxanewas stirred at rt for 1 h and the mixture concentrated in vacuum to givecompound 83 as a green solid which was dissolved in THF (5 mL) andcooled to 0° C. TEA (0.2 mL, 1.44 mmol) followed by a solution of crudeacid chloride 82 in THF (5 mL) were added and the mixture was stirred at0° C. for 20 min. The mixture was concentrated, and the residue waspurified by silica gel chromatography using a gradient of 0% to 70%Acetone in Heptanes to give 196 mg (88%) of product 84 as a yellow solid

LC-MS (Protocol B): m/z 933.5 (M+H), retention time=1.05 min. ¹H NMR(400 MHz, DMSO-d6) δ=8.26 (s, 1H), 7.94 (s, 1H), 7.46 (s, 1H), 7.39 (s,1H), 5.51 (d, J=7.8 Hz, 1H), 5.42-5.26 (m, 3H), 4.59-4.46 (m, 1H),4.33-4.23 (m, 2H), 4.23-4.15 (m, 4H), 4.15-4.07 (m, 2H), 4.04 (s, 4H),3.87 (dd, J=10.5, 18.0 Hz, 2H), 3.67 (t, J=9.8 Hz, 1H), 3.57 (t, J=10.1Hz, 1H), 3.33 (s, 1H), 3.31 (s, 1H), 2.83-2.67 (m, 2H), 2.60 (dd, J=6.4,16.2 Hz, 3H), 2.19 (s, 3H), 2.15-2.09 (m, 4H), 2.07 (s, 4H), 1.97 (s,6H).

Step 5: Synthesis of(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(((4-nitrophenoxy)carbonyl)oxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)tetrahydro-2H-pyran-3,4,5-triylTriacetate (85)

To a solution of 84 (100 mg, 0.107 mmol) in THF (5 mL) at 0° C. wasadded 4-nitrophenyl chloroformate (45 mg, 0.21 mmol), and followed byEt₃N (0.060 mL, 0.43 mmol). The mixture was stirred at 0° C. for 60 min.The mixture was concentrated in vacuum to give the crude product asyellow foam 142 mg, which was purified by reverse phase HPLC Method C)to give 50 mg (43%) of the product 85 as an off-white solid. LC-MS(Protocol B): m/z 1098.5 (M+H), retention time=1.05 min

Step 6: Synthesis of(2S,3R,4S,5S,6R)-2-(((S)-6-(5-((S)-4-(((2-((((4-((9S,12S,15S)-9-acetamido-1-(9H-fluoren-9-yl)-12-isopropyl-3,10,13-trioxo-15-(3-ureidopropyl)-2-oxa-4,11,14-triazahexadecan-16-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (86): To a solution of 85 (50 mg, 0.045 mmol) in DMF (2 mL),was added4-((9S,12S,15S)-9-acetamido-1-(9H-fluoren-9-yl)-12-isopropyl-3,10,13-trioxo-15-(3-ureidopropyl)-2-oxa-4,11,14-triazahexadecan-16-amido)benzylmethyl(2-(methylamino)ethyl)carbamate (7445 mg, 0.045 mmol) and DIPEA(0.024 mL, 0.136 mmol). The mixture was stirred at rt for 60 min, andpurified using reverse phase HPLC (Method C) to give 26 mg (31%) of theproduct 86 as a white solid: LC-MS (Protocol B): m/z 1847.1 (M+H),retention time=1.06 min ¹H NMR (400 MHz, DMSO-d6) δ=10.01 (br. s., 1H),8.25 (s, 1H), 8.20-8.12 (m, 1H), 8.09 (d, J=6.6 Hz, 1H), 8.02 (d, J=8.2Hz, 1H), 7.89 (d, J=7.8 Hz, 2H), 7.69 (d, J=7.0 Hz, 3H), 7.57 (d, J=7.4Hz, 2H), 7.47-7.37 (m, 4H), 7.37-7.30 (m, 3H), 7.26 (br. s., 3H), 5.98(br. s., 1H), 5.50 (d, J=7.0 Hz, 1H), 5.46-5.27 (m, 5H), 5.02 (d, J=19.1Hz, 2H), 4.52 (br. s., 1H), 4.39 (br. s., 1H), 4.32-4.14 (m, 13H),4.14-4.03 (m, 1H), 3.95-3.81 (m, 2H), 3.75-3.59 (m, 6H), 3.56 (br. s.,2H), 3.48 (br. s., 2H), 3.12 (br. s., 1H), 3.03 (br. s., 2H), 2.94 (s,3H), 2.97 (s, 2H), 2.87 (br. s., 2H), 2.83-2.67 (m, 3H), 2.66-2.54 (m,7H), 2.19 (s, 4H), 2.12 (s, 3H), 2.09-2.03 (m, 4H), 2.03-1.90 (m, 7H),1.85 (s, 3H), 1.78 (t, J=6.4 Hz, 1H), 1.68 (d, J=7.8 Hz, 1H), 1.61 (br.s., 2H), 1.53-1.33 (m, 5H), 1.32-1.18 (m, 2H), 0.84 (d, J=7.0 Hz, 3H),0.87 (d, J=6.6 Hz, 3H).

Step 7. Synthesis of LP:4-((S)-2-((S)-2-((S)-2-acetamido-6-aminohexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)ethane-1,2-diylbis(methylcarbamate) (77): To a solution of 86 (24 mg,0.013 mmol) in THF (2 mL) and MeOH (2 mL), at 0° C. was added a solutionof LiOH (1M, 0.26 mL, 0.26 mmol) in water and the mixture was stirred at0° C. to rt for 2 h. Acetic acid (20 uL) was added and the mixture wasconcentrated and the residue was purified by reverse phase HPLC (MethodC) to give 14 mg (69%) of the product 77 as white foam: LC-MS (ProtocolB): m/z 1456.9 (M+2H)+, retention time=0.75 min ¹H NMR (500 MHz,DMSO-d6) δ=10.07-9.96 (m, 1H), 8.29-8.18 (m, 1H), 8.18-8.09 (m, 2H),8.06 (d, J=8.1 Hz, 1H), 7.73 (d, J=8.6 Hz, 1H), 7.66 (br. s., 3H), 7.56(d, J=7.8 Hz, 2H), 7.50 (d, J=9.3 Hz, 1H), 7.47-7.39 (m, 2H), 7.35 (d,J=7.6 Hz, 1H), 7.32-7.25 (m, 2H), 6.04 (br. s., 1H), 5.11 (d, J=6.4 Hz,1H), 5.05 (br. s., 1H), 5.01 (br. s., 1H), 4.40 (br. s., 1H), 4.36-4.16(m, 10H), 4.10 (d, J=11.7 Hz, 3H), 4.00 (br. s., 3H), 3.96-3.80 (m,10H), 3.77 (br. s., 4H), 3.73-3.55 (m, 11H), 3.52 (br. s., 1H), 3.48(br. s., 3H), 3.12 (br. s., 1H), 3.05 (br. s., 2H), 3.01-2.91 (m, 4H),2.88 (br. s., 2H), 2.86-2.72 (m, 4H), 2.72-2.53 (m, 13H), 2.45 (t, J=7.5Hz, 1H), 2.06-1.92 (m, 3H), 1.86 (s, 4H), 1.71-1.56 (m, 3H), 1.56-1.42(m, 4H), 1.42-1.23 (m, 4H), 0.85 (dd, J=6.6, 14.7 Hz, 6H).

Preparation of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzylmethyl(2-(methylamino)ethyl)carbamate (96)

Step 1. Synthesis of 2,5-dioxopyrrolidin-1-yl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-valinate (88)

To a solution of compound 87 (150 g, 0.442 mol) and HOSu (56 g, 0.487mol) in THF (1800 mL) was added DCC (100 g, 0.487 mol) in portions underice-bath. After the addition, the reaction was stirred at roomtemperature overnight. The reaction was cooled to −5° C., filtered andwashed with cold THF, the filtrate was concentrated in vacuum and theresidue was re-crystallized from MTBE to give compound 88 as white solid175 g (91%).

Step 2. Synthesis of(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanoicacid (90)

To a solution of compound 89 (40.14 g, 0.229 mol) and NaHCO₃ (19.23 g,0.229 mol) in water (750 mL) was added the solution of compound 88 (100g, 0.229 mol) in DME (750 mL) dropwise under ice-bath. During theaddition, a white suspension was formed. Additional THF (400 mL) wasadded to improve the solubility. After addition, the solution wasstirred at 25-30° C. for 2 days. To the reaction was added saturated aq.K₂CO₃ to adjust pH to 8-9, then extracted with EtOAc (500 mL×5). Theaqueous layer was adjusted pH to 3-4 with aq. citric acid. A gelatinousmaterial was formed and filtered. The wet cake was dissolved in THF (1.5L). Methanol was added until the solid was dissolved. The solution wasconcentrated in vacuum to remove 30% of solvent and then cooled to roomtemperature. TBME (2 L) was added to the solution and the mixture wasstirred at room temperature overnight. The mixture was filtered and thewet cake was dried in vacuum to give compound 90 as a white solid 60 g(53%).

Step 3. Synthesis of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(92)

To the suspension of compound 90 (70 g, 0.141 mol) in DCM/MeOH (1 L/500mL) was added compound 91 (34.7 g, 0.282 mol) followed by EEDQ (69.7 g,0.282 mol). The mixture was stirred at 40° C. overnight. The reactionmixture was filtered and the wet cake was suspended in EtOAc/TBME (500mL/200 mL) and stirred for 30 min, then filtered. The solid was washedwith EtOAc/TBME to provide compound 92 as off-white solid 65 g (77%). ¹HNMR (400 MHz, DMSO-d6) δ=9.98 (s, 1H), 8.11 (d, 1H), 7.87 (d, 2H), 7.77(m, 2H), 7.52 (d, 2H), 7.39 (m, 3H), 7.30 (m, 2H), 7.21 (d, 2H), 5.97(m, 1H), 5.41 (s, 2H), 5.10 (m, 1H), 4.42 (m, 3H), 4.22 (m, 3H), 3.90(m, 1H), 2.93 (m, 2H), 1.98 (m, 1H), 1.50 (m, 2H), 1.30 (m, 2H), 0.84(m, 6H).

Step 4. Synthesis of (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(94)

To a solution of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(3.74 g, 8.31 mmol, 92) in anhydrous DMF (120 mL) was addedbis(4-nitrophenyl) carbonate (3.78 g, 12.4 mmol, 93) in portions,followed by DIPEA (1.21 g, 9.32 mmol) at 0° C. dropwise. The reactionmixture was stirred at room temperature overnight. TLC (MeOH:CH₂CH₂Cl₂=1:10) showed that the reaction was completed. The reactionmixture was added dropwise to MTBE (2.5 L) with stirring. The crudeproduct was collected by filtration. The filtrate cake was washed withMTBE and dried under high vacuum to afford compound 94 as brown solid2.7 g (57%).

Step 5. Synthesis of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyltert-butyl ethane-1,2-diylbis(methylcarbamate) (95)

To a solution of compound 94 (1.7 g, 2.217 mmol) in DMA (15 mL) wasadded HOAT (332 mg, 2.44 mmol), 2,6-lutidine (950 mg, 8.87 mmol) at 0°C. The mixture was stirred at 0° C. for 5 minutes. Then tert-butylmethyl(2-(methylamino)ethyl)carbamate (38, 459 mg, 2.44 mmol) was addedto the mixture at 0° C. and followed by DIPEA (860 mg, 6.65 mmol) at 0°C. The mixture was stirred at rt for 1 h. and poured into TBME (800 mL)and stirred for 30 min, filtered and the filter cake was concentrated invacuum to give the crude product (1.06 g) as a yellow solid. The crudeproduct was purified by silica gel chromatography (Gradient:DCM:MeOH=100:1-10:1) to give the product 95 as a white solid 600 mg(33%).

Step 6. Synthesis of4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzylmethyl(2-(methylamino)ethyl)carbamate (96)

To a stirred a solution of compound 95 (600 mg, 0.735 mmol) in dry DCM(10 mL) was added TFA (5 mL) at 0° C. and stirred at 0° C. for 1 h. Themixture was concentrated in vacuum to give the title compound 96 as awhite solid 550 mg (100%). ¹H NMR (400 MHz, DMSO-d6) δ=10.09 (br, 1H),8.40 (s, 2H), 8.14 (d, 1H), 7.90 (d, 2H), 7.74 (m, 2H), 7.61 (d, 2H),7.43 (m, 3H), 7.33 (m, 4H), 6.00 (s, 1H), 5.43 (s, 2H), 5.02 (s, 2H),4.42-4.23 (m, 4H), 3.93 (m, 1H), 3.50 (m, 2H), 3.08-2.94 (m, 4H), 2.88(s, 3H), 2.59 (m, 3H), 1.99 (m, 1H), 1.69-1.59 (m, 2H), 1.46-1.39 (m,2H), 0.90-0.85 (m, 6H).

Alternate Preparation of(2S,3S,4S,5R,6S)-6-(((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-4-(((2-((((4-((23S,26S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzyl)oxy)carbonyl)(methyl)amino)-ethyl)(methyl)carbamoyl)oxy)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicAcid (49)

Step 1: Synthesis of(2S,3R,4S,5S,6S)-2-(((S)-6-(5-((S)-4-(((2-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (97)

To a solution of compound 73 (26 mg, 0.024 mmol) in DMF (1.5 mL),Fmoc-VCPABC-DMEA linker 96 (24 mg, 0.029 mmol), 2,6-lutidine (0.0167 mL,0.14 mmol), DIPEA (0.0253 mL, 0.14 mmol) and HOAt (3.3 mg, 0.024 mmol)were added. The mixture was stirred at rt for 30 min, and concentratedin vacuum to give a residue. The crude was subjected to HPLCpurification (Method C) to give the product 97 as white solid 33 mg(83%). LC-MS (Protocol B): m/z 1660.9 (M+H), retention time=1.11 min.

Step 2: Synthesis of(2S,3S,4S,5R,6S)-6-(((S)-6-(5-((S)-4-(((2-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)-ethyl)(methyl)carbamoyl)oxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (98): To a solution of 97 (46 mg, 0.028 mmol) in THF (3 mL) andMeOH (3 mL), cooled to 0° C., was added a solution of LiOH.H₂O (12 mg,0.28 mmol) in water (0.5 mL). The mixture was stirred at 0° C. for 1 h.AcOH (0.03 mL) was added to neutralize the mixture, and concentrated invacuum to give a white solid residue. It was purified by HPLC (Method C)to give the product 98 as off-white solid 28 mg (72%). LC-MS (ProtocolB): m/z 1300.6 (M+H), retention time=0.78 min. ¹H NMR (400 MHz, DMF)δ=10.33-10.21 (m, 1H), 8.84 (d, J=7.4 Hz, 1H), 8.63 (br. s., 3H),8.41-8.28 (m, 2H), 8.05 (s, 1H), 7.81-7.63 (m, 2H), 7.57-7.41 (m, 3H),7.38 (br. s., 2H), 6.52 (br. s., 1H), 5.39 (d, J=6.6 Hz, 1H), 5.17 (br.s., 1H), 5.12 (br. s., 1H), 4.74 (br. s., 1H), 4.48-4.28 (m, 5H), 4.24(br. s., 1H), 4.09 (s, 4H), 4.04-3.89 (m, 2H), 3.84-3.67 (m, 6H),3.67-3.52 (m, 4H), 3.32 (s, 9H), 3.26 (br. s., 2H), 3.19 (br. s., 1H),3.14-3.04 (m, 4H), 3.00 (s, 2H), 2.94 (br. s., 2H), 2.85 (br. s., 2H),2.79-2.68 (m, 5H), 2.62 (s, 4H), 2.64 (s, 3H), 2.42-2.29 (m, 1H),2.23-2.05 (m, 11H), 1.92 (br. s., 1H), 1.78 (br. s., 1H), 1.60 (br. s.,2H), 1.15 (br. S., 6H).

Step 3: Synthesis of(2S,3S,4S,5R,6S)-6-(((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-4-(((2-((((4-((23S,26S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-23-isopropyl-21,24-dioxo-26-(3-ureidopropyl)-3,6,9,12,15,18-hexaoxa-22,25-diazaheptacosan-27-amido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (49): To a solution of 98 (10 mg, 0.007 mmol) was dissolved in DMF(1 mL), was added perfluorophenyl1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oate(5.6 mg, 0.009 mmol, 99) and DIPEA (0.007 mL, 0.042 mmol). The mixturewas stirred at rt for 30 min. The mixture was purified by HPLC (MethodC) to give the product 49 as white foam 10 mg (82%). LC-MS (Protocol B):m/z 1715.7 (M+H), retention time=0.89 min. 1H NMR (400 MHz,ACETONITRILE-d3) δ=8.21 (br. s., 2H), 7.56 (d, J=7.4 Hz, 1H), 7.31-7.10(m, 4H), 6.78 (s, 2H), 5.25 (br. S., 1H), 5.11 (br. s., 1H), 5.02(br.s., 1H), 4.50 (br. S., 1H), 4.18 (br. s., 2H), 3.77 (br. s., 2H),3.71 (br. s., 3H), 3.66-3.42 (m), 3.37-3.24 (m, 4H), 3.16 (br. s., 1H),3.12-2.95 (m, 4H), 2.93 (s, 2H), 2.77-2.47 (m, H), 2.22-2.02 (m, 4H),1.51 (br. s., 2H), 0.95 (br. S., 6H).

Preparation of4-((S)-2-((S)-2-((S)-2-acetamido-6-aminohexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)ethane-1,2-diylbis(methylcarbamate) (100)

Step 1: Synthesis of4-((S)-2-((S)-2-((S)-2-acetamido-6-aminohexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-8-(chloromethyl)-6-(5-((S)-8-(chloromethyl)-1-methyl-4-(phosphonooxy)-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl)-5-oxopentanoyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl)ethane-1,2-diylbis(methylcarbamate) (100): To a solution of 55 (35 mg,0.041 mmol) in DMF (3 mL), was added 74 (45 mg, 0.045 mmol), lutidine(0.019 mL, 0.16 mmol), DIPEA (0.029 mL, 0.16 mmol) and HOAt (5.6 mg,0.041 mmol). The mixture was stirred at rt for 3 h. To the resultingmixture, piperidine (0.3 mL, 3 mmol) was stirred at rt for 20 min. Themixture was concentrated in vacuum, and the residue was purified by HPLC(Method C) to give the product 100 as white powder after freeze dry 35mg (62%). LC-MS (Protocol B): m/z 1374.8 (M+H), retention time=0.92 min.¹H NMR (400 MHz, DMSO-d6) δ=8.45 (d, J=13.7 Hz, 1H), 8.14 (s, 1H), 8.17(s, 1H), 8.05 (d, J=7.4 Hz, 1H), 7.76 (br. s., 3H), 7.58 (d, J=8.2 Hz,1H), 7.52-7.39 (m, 2H), 7.30-7.16 (m, 1H), 6.09 (br. s., 1H), 5.15-4.94(m, 2H), 4.40 (br. s., 1H), 4.35-4.08 (m, 8H), 3.95-3.79 (m, 2H), 3.69(br. s., 1H), 3.60 (d, J=10.5 Hz, 3H), 3.49 (br. s., 3H), 3.13 (br. s.,1H), 3.06 (br. s., 1H), 3.03-2.91 (m, 4H), 2.88 (s, 2H), 2.76 (br. s.,4H), 2.61 (d, J=6.6 Hz, 2H), 2.56 (s, 6H), 1.98 (m, 3H), 1.86 (s, 3H),1.65 (m, 3H), 1.58-1.49 (m, 3H), 1.46 (m, 2H), 1.40-1.20 (m, 3H), 0.85(m, 6H).

As noted herein, the described compounds in Tables 1 and 2 may exist intheir acetate or phenol prodrug stage, which both rapidly convert intothe bis cyclopropyl active drug species. This interconversion occursrapidly in the assay medium. Hence, all three compound forms arefunctionally equivalent and result in the same growth inhibitory valuesin cancer cell proliferation assays. Thus describing compounds as“bis-acetates” also entails the description as their functionallyequivalent phenol- and bis cyclopropyl species.

The following payloads were prepared. All payloads were prepared eitherusing the general procedures A and B or by indicated methods

TABLE 1 Examples of Payloads LC-MS Retention Compound time m/z StructureID (Method) [M + H]+

24 1.11 min (Protocol B) 687.2

 7 1.12 min (Protocol B) 711.1

26 1.11 min (Protocol B) 687.2

12 1.12 min (Protocol B) 711.1

17 1.12 min (Protocol B) 711

25 1.11 min (Protocol B) 687.1

60 2.39 min (Protocol A) 747

61 0.93 min (Protocol B) 683

57  0.9 min (Protocol B) 779

33 1.86 min (Protocol A) 872

59 0.74 min (Protocol B) 893

87 2.25 min (Protocol A) 689.3

88 2.25 min (Protocol A) 691.3

89 2.25 min (Protocol A) 693.3

TABLE 2 The following payloads are prepared using the general syntheticprocedures A and B. Structure

In the above examples, the variable Pg is H, acyl, phosphate PO₃H₂, acarbohydrate, an amino acid or a peptide (and in particular a peptidethat is cleaved by proteases, such as cathepsins and matrixmetalloproteinases).

The following linker/payloads were prepared:

TABLE 3 Prepared Linker-Payloads LC-MS Observed Retention Ion inCompound time Mass. Structure ID (Method) Spec.

41 1.61 minutes (Protocol A) 1642 [M + 2H]

49 0.89 minutes (Protocol B) 1714 [M + 2H]

56 0.95 minutes (Protocol B) 1618 [M + 2H]

62 2.01 min (Protocol A) 1026 [M + 2H]

76 0.75 min (Protocol B) 1471.1 (M + 2H)⁺

77 0.75 min (Protocol B) 1456.9 (M + 2H)⁺

100  0.92 min (Protocol B) 1374.8 (M + 2H)⁺

Table 4: The following linker/payloads are prepared using the proceduredescribed above for preparation of linker/payload analogs shown in Table3.

TABLE 4 Additional Linker/Payloads Linker Structures for Table 4

where P represents the point of attachment to said payload, each R⁷ isindependently H or —C₁-C₂₀ alkyl, R⁸ is —C₁-C₂₀ alkyl, —C₆-C₁₄ aryl or—C₆-C₁₄ heteroaryl, n = 0-20, and m = 0-20.

The attachment points of the above linker structures are indicated andare located at the payload phenol:

Payload Structure (P)

In Table 4, Pg is H, acyl, phosphate PO₃H₂, a carbohydrate, an aminoacid or a peptide (in particular a peptide that is cleaved by proteases,such as cathepsins and matrix metalloproteinases).

TABLE 5 Additional Linker-Payloads Linker Structures for Table 5 from:

where P represents the point of attachment to said payload, each R⁷ isindependently H or —C₁-C₂₀ alkyl, R⁸ is —C₁-C₂₀ alkyl, —C₆-C₁₄ aryl or—C₆-C₁₄ heteroaryl, n = 0-20, and m = 0-20.

The attachment points of the above linker structures are located at theaniline N function:

In Table 5, Pg is H, acyl, phosphate POH, a carbohydrate, an amino acidor a peptide (in particular a peptide that is cleaved by proteases, suchas cathepsins and matrix metalloproteinases).Experimental Procedures for Biological Assessment of Payloads andAntibody Drug ConjugatesCell Lines for Payload Viability Assays

Cancer cell lines were obtained from ATCC (Manassas, Va.). N87 (humangastric carcinoma derived from metastatic liver site). HL60 (leukemia)and MDA-MB-361 DYT2 (human breast carcinoma MDA-MB-361) were grown inRPMI 1640 media. All media were supplemented with 10% fetal bovineserum, 1% sodium pyruvate, and 1% L-glutamine (Invitrogen, Grand Island,N.Y.). Human umbilical vein endothelial cells (HUVEC) were obtained fromLonza (Allendale, N.J.) and maintained in EGM2 media supplemented withEGM-2 SingleQuots (Lonza #CC-4176). All cells were maintained in ahumidified incubator (37° C., 5% CO₂).

Cytotoxicity Assay Procedure for Payloads

Cells in 100 μl medium were cultured in a 96-well plate. Cancer celllines treated with the indicated compounds by adding 50 μl of 3× stocksin duplicate at 10 concentrations. Cells were incubated with compoundsfor four days, then 30 μl of CellTiter® 96 AQueous One MTS Solution(Promega Cat #G3582) was added to the cells, incubated 1.5 hr at 37° C.,then absorbance measured at 490 nm on a Victor plate reader (PerkinElmer, Waltham, Mass.). Relative cell viability was determined as apercentage of untreated control wells. IC50 values were calculated usingfour parameter logistic model #203 with XLfit v4.2 (IDBS, Guildford,Surry, UK).

Cytotoxicity Assay Procedure for Anti-IL13Rα2 ADCs

Cell lines used: A375 (melanoma), PC3MM2 (prostate), PC3 (prostate)

Cells were seeded into 96-well plate overnight before expose toincreasing concentrations of anti-IL13Rα2 ADCs or control ADCs. Afterfour days, viability of each culture was assessed against control cells.IC₅₀ values were calculated by logistic non-linear regression.

Cytotoxcity Assays Procedure for Anti-CD33 ADCs

Human AML cell lines (HL60, NB4, HEL92.1.7, and Raji) were obtained fromAmerican Type Culture Collection (Manassas, Va.). The cells were grownin RPMI media, supplemented with 10% FBS, 1% Pen/Strep and L-GlutamineMixture, 1 mM sodium pyruvate, 10 mM HEPES, and 0.27% glucose (LifeTechnology, Carlsbad, Calif.). Cell lines were tested for theirendogenous activity levels of P-glycoprotein, multidrug resistanceprotein, and breast cancer resistance protein were determined using acommercial flow cytometric kit (eFLUXX-ID; ENZO Life Sciences, PlymouthMeeting, Pa.).

For cytotox assays, cells were plated in a 96-well opaque plates(Corning) and treated with varying concentrations of compounds for 4days. Viability was determined by using a CellTiter Glo luminescent cellviability assay kit (Promega, Madison, Wis.) and measured using a VictorX3 plate reader (Perkin Elmer Waltham, Mass.). The data were normalizedto the control group (DMSO or PBS). IC₅₀ values were defined as theconcentration that causes 50% growth inhibition. IC₅₀ values werecalculated using a logistic nonlinear regression, model no. 203 with XLfit v4.2 (IDBS, Guldford, Surry, UK). All experimental points were setupin two replicate wells and independently performed in duplicate.

Cytotoxcity Assays for Anti-Trastuzumab ADCs

Target expressing N87 (gastric cancer), MDA-MB-361-DYT2 (breast cancer))or non-expressing (HT-29) cells were seeded in 96-well cell cultureplates for 24 hours before treatment. Cells were treated with3-foldserially diluted antibody-drug conjugates Cell viability wasdetermined by CellTiter 96® AQ_(ueous) One Solution Cell ProliferationMTS Assay (Promega, Madison Wis.) 96 hours after treatment. Relativecell viability was determined as percentage of untreated control. IC₅₀values were calculated using a four parameter logistic model #203 withXLfit v4. 2 (IDBS, Guildford, Surry, UK).

Results of cytotoxicity of payloads and ADCs are shown in Tables 6 and10

TABLE 6 Exemplifeid Free Payload Potencies: Compound ID N87 [nM] HL60[nM] DYT2 [nM] 24 0.024 <0.005 0.171 7 0.022 <0.005 0.185 26 0.263 0.0141.670 12 2.258 0.180 5.077 17 1.296 0.084 6.062 25 41.600 NA >100 600.122 0.009 0.079 61 13.646 0.806 28.587 57 11.020 0.873 37.588 87 0.0360.349 0.002 88 0.041 0.41 0.004 89 0.041 0.302 0.002

Exemplification of Antibody Drug Conjugates

Method D: General Procedure for Conjugation of Antibody withLinker-Payload Via Internal Disulfides

A solution of therapeutic antibody in Dulbecco's Phosphate BufferedSaline (PBS, Lonza, pH 7.4) was reduced by addition of 3.0-3.5equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 mMsolution in PBS). The reaction was incubated at 37° C. for 1-2 h andthen allowed to cool to ambient temperature. Conjugation was performedby addition of 7 equivalents of linker-payload [10 mM solution inN,N-dimethylacetamide (DMA)]. Additional DMA was added to reactionmixture to achieve 15% (v/v) total organic solvent component in finalreaction mixture. The reaction was incubated for 1 h at ambienttemperature. After 1 h at ambient temperature, crude reaction was eitherbuffer exchanged PBS via GE Sephadex desalting columns or the excesslinker-payload was quenched via addition of 10 equivalents of cysteine(20 mM solution in PBS). In either case, crude material was thenpurified by size exclusion chromatography (SEC, Protocol C). Themonomeric fractions were pooled and concentrated if necessary to givethe ADC.

Method E: Site-Specific Conjugation of Linker-Payloads to a TrastuzumabAntibody Containing Engineered Cysteine Residues

A solution of therapeutic antibody containing an engineered cysteineresidue (Kabat numbering, see WO2013093809) was prepared in 50 mMphosphate buffer, pH 7.4. PBS, EDTA (0.5 M stock), and TCEP (0.5 Mstock) were added such that the final protein concentration was ˜10mg/mL, the final EDTA concentration was ˜20 mM, and the final TCEPconcentration was approximately ˜6.6 mM (100 molar eq.). The reactionwas allowed to stand at rt for 2-48 h and then buffer exchanged into PBSusing GE PD-10 Sephadex G25 columns per the manufacturer's instructions.Alternative methods such as diafiltration or dialysis are also useful inparticular circumstances. The resulting solution was treated withapproximately 50 equivalents of dehydroascorbate (50 mM stock in 1:1EtOH/water). The antibody was allowed to stand at 4° C. overnight andsubsequently buffer exchanged into PBS using GE PD-10 Sephadex G25columns per the manufacturer's instructions. Again, alternative methodssuch as diafiltration or dialysis are also useful in particularcircumstances.

Conjugation to the antibody thus prepared was performed by addition of10 equivalents of linker-payload [10 mM solution inN,N-dimethylacetamide (DMA)]. Additional DMA was added to reactionmixture to achieve 15% (v/v) total organic solvent component in finalreaction mixture. In some instances, additional PBS was added to achievefinal total antibody concentration in the reaction of 5-10 mg/mL. After1-2 h at rt, the reaction mixture was buffer exchanged into PBS (perabove) and purified by size exclusion chromatography (SEC, Protocol D).The monomeric fractions were pooled and concentrated if necessary togive the ADC. In some instances, the ADC was then subsequently incubatedafter SEC purification with 0.5-1.0 g Bio-Beads SM-2 adsorbent (Bio-Rad)at 23-37 C for 4-18 h, filtered, and then concentrated if required.

Method F: Enzyme Mediated Conjugation to Antibody Carrying ReactiveGlutamine Residues:

Therapeutic antibody carrying transglutamine enzyme-reactive glutamineresidues was dialyzed into sterile water (Lonza). The transglutaminasemediated conjugation was carried out by mixing 5.0-15 mg/mLtransglutaminase reactive glutamine containing antibody in water with 30mM Potassium Phosphate pH7.4 DILUT-IT Dissolution Media Concentrate (J.T. Baker), 150 mM sodium chloride (NaCl), 15.0-20.0-fold molar excess ofamino alkyl linker carrying payload (10-30 mM solution in DMSO), and30-60 mg/mL transglutaminase enzyme powder (Ajinomoto Activa TI).Additionally, DMSO was added to reaction mixture to achieve 5-10% (v/v)total organic solvent component in final reaction mixture. In someinstances, additional water was added to achieve final total antibodyconcentration in the reaction of 5-10 mg/mL. The resulting mixture wasincubated at ambient temperature for 5-6 hours. Next, another 30-60mg/mL of transglutaminase solid was added and the mixture furtherincubated at ambient temperature for an additional 18 hours. Afterovernight, the crude reaction was buffer exchanged into Dulbecco'sphosphate buffered saline (PBS, pH7.4, Lonza) using GE HealthcareSephadex buffer exchange columns per manufacturer's instructions. Crudematerial was purified either by size exclusion chromatography (SEC,Protocol D) or hydrophobic interaction chromatography (HIC, Protocol G).After purification, the monomeric fractions were pooled and concentratedif necessary to give the ADC. In examples where ADC was purification byHIC, the pooled fractions were then buffer exchanged into Dulbecco'sphosphate buffered saline (PBS, pH7.4, Lonza) using GE HealthcareSephadex buffer exchange columns per manufacturer's instructions. Insome instances, the ADC was then subsequently incubated afterpurification with 0.5-1.0 g Bio-Beads SM-2 adsorbent (Bio-Rad) at 23-37C for 4-18 h, filtered, and then concentrated if required.

The ADC was characterized via size exclusion chromatography (SEC) andhydrophobic interaction chromatography (HIC) for purity, and liquidchromatography electrospray ionization tandem mass spectrometry (LC-ESIMS) to calculate drug-antibody ratio (DAR, loading).

Protocol C: Column: Agilent Poroshell 300SB-C8, 75×2.1 mm, 2.6 μm;Mobile phase A:0.1% formic acid in water (v/v); Mobile phase B: 0.1%formic acid in acetonitrile (v/v); Gradient: Initial Conditions: 20% Bto 45% B over 4 minutes; Flow rate: 1.0 mL/minute. Temperature: 60° C.;Detection: 220 nm; MS (+) range 400-2000 Da; Injection volume: 10 μL;Instrument: Agilent 1100 LC, Waters MicromassZQ MS. Deconvolution wasperformed using MaxEnt1.

Protocol D: Column: GE Superdex 200 (10/300 GL); Mobile phase: Phosphatebuffered saline (PBS, 1×, pH 7.4); Isocratic; Flow rate: 1.0 mL/minute.Temperature: room temperature; Instrument: GE Akta Explorer.

Protocol E: Column=Waters BEH300-C4, 2.1×100 mm (P/N═186004496);Instrument=Acquity UPLC with an SQD2 mass spec detector; Flow rate=0.7mL/min; Temperature=80° C.; Buffer A=water+0.1% formic acid; BufferB=acetonitrile+0.1% formic acid. The gradient runs from 3% B to 95% Bover 2 minutes, holds at 95% B for 0.75 min, and then re-equilibrates at3% B. The sample is reduced with TCEP or DTT immediately prior toinjection. The eluate is monitored by LCMS (400-2000 daltons) and theprotein peak is deconvoluted using MaxEnt1. DAR is reported as a weightaverage loading as has been previously described.

Protocol F: Column: TSKGel Butyl NPR, 4.6 mm×3.5 cm (P/N═S0557-835);Buffer A=1.5 M ammonium sulfate containing 10 mM phosphate, pH 7; BufferB=10 mM phosphate, pH 7+20% isopropyl alcohol; Flow rate=0.8 mL/min;Temperature=ambient; Gradient=0% B to 100% B over 12 minutes, hold at100% B for 2 minutes, then re-equilibrate at 100% A; Instrument: Agilent1100 HPLC.

Protocol G: Column: GE HiTrap Butyl HP, 5 mL; Mobile phases: 1Mpotassium phosphate, 50 mM Tris-HCl, pH7 (Buffer A); 50 mM Tris-HCl, pH7(Buffer B); Gradient elution: 0-100% Buffer B over 10-20 column volumes;Flow rate: 5.0 mL/minute. Temperature: room temperature; Instrument: GEAkta Explorer.

TABLE 7 Structure of ADC and Payload Linkers used to prepare them ADC#Structure LP used for synthesis ADC#1

41 ADC#2

76 ADC#3

77 ADC#4

41 ADC#5

41 ADC#6

41

X=In the above table, “X” indicates the antibody used for conjugation asdenoted in Table 5.

TABLE 8 General Method for Preparation of ADCs General Linker- Methodfor payload Theoretical ADC# preparation Antibody used used MW(increase) ADC#1 D IL13Rα2-AB08-v1.0 41 1640 ADC#2 FTrastuzumab-H16-K222R- 76 1450 hG1 ADC#3 F Trastuzumab-H16-K222R- 771436 hG1 ADC#4 E CD33-11A1-v1417-hG1-(C) 41 1640 ADC#5 ECD33-11A1-v1417-K334C- 41 1640 K392C-hG1-(C334 + C392) ADC#6 ECD33-11A1-v1417-K334C- 41 1640 hG1-(C334)

TABLE 9 Purification method and Analytical Characterization of ADCs Drugper Drug per Observed Antibody Antibody □ mass ratio ratio HIC for the(DAR) (DAR) retention Heavy (LC/MS (HIC Iso- time Chain Method) Method)Purification lated (Method (HC) (protocol (Protocol ADC# method yield F)portion E) F) ADC#1 Protocol D 18% NA 1642 2.1 NA ADC#2 Protocol D 36%5.43 1450 1.5 1.7 ADC#3 Protocol D 34% 5.48 1436 1.8 1.9 ADC#4 ProtocolD 52% 6.20 1641 2.8 2.6 ADC#5 Protocol D 72% 5.34 1639 3.6 3.5 ADC#6Protocol D 73% 5.41 1640 2.0 2.0

TABLE 10 In vitro Cytotoxicity data for ADCs #1-6 in Antigen +ve andAntigen −ve cell lines MDA-MB- HEL N87 361- HT29 A375 PC3MM2 PC3 HL-6092.1.7 Raji ADC# (+ve) DYT2 (−ve) (+ve) (+ve) (−ve) (+ve) (+ve) (−ve)ADC#1 0.9 0.5 >1000 ADC#2 0.6 >470 >470 ADC#3 0.4 >470 >470 ADC#4 1.7<0.5 3040.7 ADC#5 1.5 0.6 >10,000 ADC#6 2.1 0.6 >10,000

The following ADC's are prepared, including the linker/payloads shown inTables 4 and 5 conjugated to antibodies of interest using theappropriate conjugation method:

We claim:
 1. A method of treating cancer comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof Formula (IIIA):AB-(L-P)₁₋₂₀  (IIIA) or a pharmaceutically acceptable salt or solvatethereof, wherein: AB is an antibody: P is:F¹-L¹-T-L²-F² wherein: F¹ and F² are each independently selected fromring systems A, B, C and D:

where: each R is independently selected from the group consisting of H,—C₁-C₂₀ alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, halo, deuterium,hydroxyl, alkoxy, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —NO₂,—C₆-C₁₄ aryl and —C₆-C₁₄ heteroaryl, or wherein two or more R optionallyjoin to form a ring or rings, and wherein said —C₆-C₁₄ aryl and —C₆-C₁₄heteroaryl are optionally substituted with 1 to 5 substituentsindependently selected from —C₁-C₁₀ alkyl, —C₁-C₁₀ alkoxy, -halo,—C₁-C₁₀ alkylthio, -trifluoromethyl, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈alkyl)₂, —C₁-C₁₀ alkyl-N(C₁-C₈ alkyl)₂, —C₁-C₃ alkylthio, —NO₂ or—C₁-C₁₀ heterocyclyl, for each ring system in which R appears: each V¹is independently a bond, O, N(R) or S, for each ring system in which V¹appears: each V² is independently O, N(R) or S, for each ring system inwhich V² appears: W¹ and W² are each independently H, —C₁-C₅ alkyl,-phenyl, —C(O)OR, —C(O)SR, —C(O)NHN(R)₂ or —C(O)N(R)₂ for each ringsystem in which W¹ and W² appear; each X is independently selected from—OH, —O-acyl, azido, halo, cyanate, thiocyanate, isocyanate,thioisocyanate, or

for each ring system in which X appears; each Y is independentlyselected from a bond, H, —C(O)R^(A), —C(S)R^(A), —C(O)OR^(A),—S(O)₂OR^(A), —C(O)N(R^(A))₂, —C(S)N(R^(A))₂, glycosyl, —NO₂,—P(O)(OR^(A))₂, an amino acid and a peptide for each ring system inwhich Y appears, wherein each R^(A) is independently selected from H,—C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl, —C₁-C₂₀ alkylN(R)₂, —C₁-C₂₀ alkylene,—C₁-C₈ heteroalkylene, —C₆-C₁₄ arylene, aralkylene, —C₁-C₁₀ heterocyclo,—C₃-C₈ carbocyclo and —C₁-C₂₀ alkylN(R)—, and R^(F) where said R^(A) isoptionally substituted with 1 to 3 substituents independently selectedfrom R, and wherein at least one Y-containing Ring System is present andis divalent and is bonded to L, R^(F) is —N(R⁶)QN(R⁵)C(O)— and is bondedto L at the carbonyl adjacent N(R⁵), wherein R⁵ and R⁶ are eachindependently selected from the group consisting of H, —C₁-C₈ alkyl,—C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl and—C₃-C₈ carbocyclyl, or R⁵ or R⁶ loins with a substituted carbon on Q toform a —C₁-C₁₀ heterocyclic or —C₆-C₁₄ heteroaryl ring, or R⁵ and R⁶join together to form a —C₁-C₁₀ heterocyclic or —C₆-C₁₄ heteroaryl ringsystem, and where Q is —C₁-C₈ alkylene-, —C₁-C₈ heteroalkylene-, —C₆-C₁₄arylene-, -aralkylene-, —C₁-C₁₀ heterocyclo- or —C₃-C₈ carbocyclo-,wherein Q, R⁵ and R⁶ are each independently optionally substituted with1 to 3 substituents independently selected from R: each Z isindependently selected from the group consisting of H, —C₁-C₈ alkyl,—C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo, and wherein said C₁-C₈ alkyl, —C₁-C₈heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈ alkyl)₂, —C(O)OH,—C(O)NHNH₂ and —C(O)-halo are each optionally substituted with 1 to 3substitutents independently selected from R, for each ring system inwhich Z appears: L¹ and L² are each independently selected from a directbond T is —C(A¹)X¹-T²-X¹C(B¹)—, where T² is:

wherein each X¹ is independently a bond, —NR^(E)—, —O— or —S—, whereinA¹ and B¹ are each independently ═O or ═S, wherein R¹, R², R³, and R⁴are each independently R^(E) or R¹ and R² form a ring system, or R³ andR⁴ form a ring system, or both R¹ and R², and R³ and R⁴, eachindependently form ring systems, or R¹ and R³ form a ring system, or R²and R⁴ form a ring system, or both R¹ and R³, and R² and R⁴, eachindependently form ring systems, where said ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₈ carbocyclycl,or R¹, R², R³ and R⁴ are each bonds to different carbons on D, wherein qand j are each independently an integer from 0 to 50 and m is an integerfrom 1 to 50, and wherein D is selected from the group consisting of—C₁-C₁₀ heterocyclo and —C₃-C₈ carbocyclo, where said —C₁-C₁₀heterocyclo and —C₃-C₈ carbocyclo are optionally substituted with—R^(E), —C(O)R^(E), —C(O)OR^(E), —N(R^(E))₂, —N(R)C(O)R^(E) or—N(R)C(O)OR^(E), and D is additionally optionally substituted by 1 to 2R, wherein each R^(E) is independently selected from the groupconsisting of H, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, -aryl, -aralkyl,—C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl,—C(O)N(C₁-C₈ alkyl)₂, and —C(O)-halo, and wherein each R^(E) isoptionally substituted with 1 to 3 substitutents independently selectedfrom R; L is L^(A)-L^(B)-(L^(C))₁₋₃ wherein an L^(C) is bound to Y;L^(A) is selected from: a bond to AB, —NR-(bond to AB),-heteroaryl-(bond to AB),

L^(B) is L^(B1)-L^(B2)-L^(B3) wherein L^(B1) is absent or is one or morecomponents selected from the grouj consisting of —C(O)—, —C(S)—,—C(O)NR—, —C(O)C₁-C₆alkyl-, —C(O)NRC₁-C₆alkyl-,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C(O)C₁-C₆alkylNRC(O)—,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆-C(O)—,—C₁-C₆alkyl-S—S—C₁-C₆alkylNRC(O)CH₂—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆NRC(O)CH₂—,—C(O)C₁-C₆alkyl-NRC(O)C₁₋₆alkyl-, —N═CR-phenyl-O—C₁-C₆alkyl-,—N═CR-phenyl-O—C₁-C₆alkyl-C(O)—, —C(O)—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NRC(O)—,—C(O)C₁-C₆alkyl-phenyl(NR—C(O)C₁-C₆alkyl)₁₋₄-,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—NRC(O)C₁-C₆alkyl-, —C₁-C₆alkyl-, —S—,—C(O)—CH(NR—C(O)C₁-C₆alkyl)-C₁-C₆alkyl- and (—CH₂—CH₂—O—)₁₋₂₀; L^(B2) isAA₀₋₁₂, wherein AA is a natural amino acid- or a non-natural amino acid;L^(B3) is p-aminobenzoic acid, p-aminobenzyloxycarbonyl,—C(O)(CH₂)₀₋₅₀C(O)— or absent, L^(C) is absent or is independentlyselected from the group consisting of —C₁-C₆alkylene-,—NRC₃-C₈-heterocyclylNR—, —NRC₃-C₃-carbocyclylNR—, —NRC₁-C₆alkylNR—,—NRC₁-C₆alkylene-, —S—, —NR—, —NRNR—, —O(CR₂)₁₋₄S—S(CR₂)₁₋₄N(R)—,—NRC₁-C₆-alkylenephenyleneNR—, —NRC₁-C₆alkylenephenyleneSO₂NR—,—OC₁-C₆alkylS-SC₁-C₆alkylC(COOR)NR—,

wherein X^(A) is CR or N, X^(B) is CH, CR(C(R)₂)₁₋₃NR, CR(C(R)₂)₁₋₃₀,CR(C(R)₂)₁₋₃C(O)NR, CR—(C(R)₂)₁₋₃C(O)NRNR, CR(C(R)₂)₁₋₃SO₂NR,CR(C(R)₂)₁₋₃NRNR, CR(C(R)₂)₁₋₃NRC(O) or N, each X^(C) is R: each X^(D)is —(CH₂)₁₋₅—, or is absent; X^(E) is O, S, C(R)₂, C(R)(C(R)₂)₁₋₃—NR₂ orNR, and each X^(F) is (C(R)₂)₁₋₃—NR or C(R)₂—(C(R)₂)₁₋₃—O.
 2. A methodof treating cancer comprising administering to a patient in need thereofa therapeutically effective amount of a compound of Formula (IIIB):

or a pharmaceutically acceptable salt or solvate thereof, wherein: AB isan antibody; F¹ and F² are each independently selected from ring systemsA, B, C and D:

where: each R is independently selected from the group consisting of H,—C₁-C₂₀ alkyl, —C₂-C₆ alkenyl, —C₂-C₆ alkynyl, halo, deuterium,hydroxyl, alkoxy, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈ alkyl)₂, —NO₂,—C₆-C₁₄ aryl and —C₆-C₁₄ heteroaryl, or wherein two or more R optionallyjoin to form a ring or rings, and wherein said —C₆-C₁₄ aryl and —C₆-C₁₄heteroaryl are optionally substituted with 1 to 5 substituentsindependently selected from —C₁-C₁₀ alkyl, —C₁-C₁₀ alkoxy, -halo,—C₁-C₁₀ alkylthio, -trifluoromethyl, —NH₂, —NH(C₁-C₈ alkyl), —N(C₁-C₈alkyl)₂, —C₁-C₁₀ alkyl-N(C₁-C₈ alkyl)₂, —C₁-C₃ alkylthio, —NO₂ or—C₁-C₁₀ heterocyclyl, for each ring system in which R appears: each V¹is independently a bond, O, N(R) or S, for each ring system in which V¹appears; each V² is independently O, N(R) or S, for each ring system inwhich V² appears; W¹ and W² are each independently H, —C₁-C₅ alkyl,-phenyl, —C(O)OR, —C(O)SR, —C(O)NHN(R)₂ or —C(O)N(R)₂ for each ringsystem in which W¹ and W² appear; each X is independently —OH, —O-acyl,azido, halo, cyanate, thiocyanate, isocyanate, thioisocyanate, or

for each ring system in which X appears: each Y is independentlyselected from the group consisting of H, —C₁-C₆ alkyl-R^(A) —C(O)R^(A),—C(S)R^(A), —C(O)OR^(A), —S(O)₂OR^(A), —C(O)N(R^(A))₂, —C(S)N(R^(A))₂,glycosyl, —NO, —PO(OR^(A))₂, an amino acid and a peptide for each ringsystem in which Y appears, wherein each R^(A) is independently selectedfrom the group consisting of H, —C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl,—C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclyl and—C₁-C₂₀ alkylN(R)₂, wherein said —C₁-C₂₀ alkyl, —C₁-C₈ heteroalkyl,—C₆-C₁₄ aryl, aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclyl and—C₁-C₂₀ alkylN(R)₂ are optionally substituted with 1 to 3 substitutentsindependently selected from R; each Z is independently selected from thegroup consisting of H, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl,-aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl,—C(O)N(C₁-C₈ alkyl)₂, —C(O)OH, —C(O)NHNH₂ and —C(O)-halo, and whereinsaid C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, —C₆-C₁₄ aryl, -aralkyl, —C₁-C₁₀heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl, —C(O)N(C₁-C₈alkyl)₂, —C(O)OH, —C(O)NHNH₂ and —C(O)-halo are each optionallysubstituted with 1 to 3 substitutents independently selected from R, foreach ring system in which Z appears; L¹ and L² are each independentlyselected from a direct bond T is: C(A¹)X¹-T²-X¹C(B¹)—, where T² is:

wherein each X¹ is independently a bond, —NR^(E)—, —O— or —S—, whereinA¹ and B¹ are each independently ═O or ═S, wherein R¹, R², R³, and R⁴are each independently R^(E) or R¹ and R² form a ring system, or R³ andR⁴ form a ring system, or both R¹ and R², and R³ and R⁴ eachindependently form ring systems, or R¹ and R³ form a ring system, or R²and R⁴ form a ring system, or both R¹ and R³, and R² and R⁴ eachindependently form ring systems, where the ring systems areindependently selected from —C₁-C₁₀ heterocyclyl or —C₃-C₈ carbocyclycl,or R¹, R², R³ and R⁴ are each bonds to different carbons on D, wherein qand j are each independently an integer from 0 to 50 and m is an integerfrom 1 to 50, and wherein D is selected from the group consisting of—C₁-C₁₀ heterocyclo and —C₃-C₈ carbocyclo, where said —C₁-C₁₀heterocyclo and —C₃-C₈ carbocyclo are substituted with one member of thegroup selected from N(R^(E))C(O)— where the carbonyl is bonded to L, and—C(O)— where the carbonyl is bonded to L, and additionally optionallysubstituted by 1 to 2 R; where each R^(E) is independently selected fromthe group consisting of H, —C₁-C₈ alkyl, —C₁-C₈ heteroalkyl, -aryl,-aralkyl, —C₁-C₁₀ heterocyclyl, —C₃-C₈ carbocyclyl, —C(O)OC₁-C₈ alkyl,—C(O)N(C₁-C₈ alkyl)₂, and —C(O)-halo, and wherein each R^(E) isoptionally substituted with 1 to 3 substitutents independently selectedfrom R: L is L^(A)-L^(B)-(L^(C))₁₋₃ wherein an L^(C) is bound to T²;L^(A) is selected from: a bond to AB, —NR-(bond to AB),-heteroaryl-(bond to AB),

L^(B) is L^(B1)-L^(B2)-L^(B3) wherein L^(B1) is absent or is one or morecomponents selected from the group consisting of —C(O)—, —C(S)—,—C(O)NR—, —C(O)C₁-C₆alkyl-, —C(O)NRC₁-C₆alkyl-,—C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C(O)C₁-C₆alkylNRC(O)—,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆-C(O)—,—C₁-C₆alkyl-S—S—C₁-C₆alkylNRC(O)CH₂—, —C₁-C₆alkyl(OCH₂CH₂)₁₋₆NRC(O)CH₂—,—C(O)C₁-C₆alkyl-NRC(O)C₁₋₆alkyl-, —N═CR-phenyl-O—C₁-C₆alkyl-,—N═CR-phenyl-O—C₁-C₆alkyl-C(O)—, —C(O)—C₁-C₆alkyl(OCH₂CH₂)₁₋₆NRC(O)—,—C(O)C₁-C₆alkyl-phenyl(NR—C(O)C₁-C₆alkyl)₁₋₄-,—C(O)C₁-C₆alkyl(OCH₂CH₂)₁₋₆—NRC(O)C₁-C₆alkyl-, —C₁-C₆alkyl-, —S—,—C(O)—CH(NR—C(O)C₁-C₆alkyl)-C₁-C₆alkyl- and (—CH₂—CH₂—O—)₁₋₂₀: L^(B2) isAA₀₋₁₂, wherein AA is a natural amino acid- or a non-natural amino acid;L^(B3) is p-aminobenzoic acid, p-aminobenzyloxycarbonyl,—C(O)(CH₂)₀₋₅₀C(O)— or absent; L^(C) is absent or is independentlyselected from the group consisting of —C₁-C₆alkylene-,—NRC₃-C₃-heterocyclylNR—, —NRC₃-C₃-carbocyclylNR—, —NRC₁-C₆alkylNR—,—NRC₁-C₆alkylene-, —S—, —NR—, —NRNR—, —O(CR₂)₁₋₄S—S(CR₂)₁₋₄N(R)—,—NRC₁-C₆-alkylenephenyleneNR—, —NRC₁-C₆alkylenephenyleneSO₂NR—,—OC₁-C₆alkylS-SC₁-C₆alkylC(COOR)NR—,—NRC(COOR)C₁-C₆alkylS-SC₁-C₆alkylO—,

wherein X^(A) is CR or N, X^(B) is CH, CR(C(R)₂)₁₋₃NR, CR(C(R)₂)₁₋₃O,CR(C(R)₂)₁₋₃C(O)NR, CR—(C(R)₂)₁₋₃C(O)NRNR, CR(C(R)₂)₁₋₃SO₂NR,CR(C(R)₂)₁₋₃NRNR, CR(C(R)₂)₁₋₃NRC(O) or N; each X^(C) is R; each X^(D)is —(CH₂)₁₋₅—, or is absent; X^(E) is O, S, C(R)₂, C(R)(C(R)₂)₁₋₃—NR₂ orNR, and each X^(F) is (C(R)₂)₁₋₃—NR or C(R)₂—(C(R)₂)₁₋₃—O.
 3. The methodof claim 1 wherein said cancer is bladder cancer, breast cancer,cervical cancer, colon cancer, endometrial cancer, kidney cancer, lungcancer, esophageal cancer, ovarian cancer, prostate cancer, pancreaticcancer, skin cancer, stomach (gastric) cancer, testicular cancer,leukemias or lymphomas.
 4. The method of claim 2 wherein said cancer isbladder cancer, breast cancer, cervical cancer, colon cancer,endometrial cancer, kidney cancer, lung cancer, esophageal cancer,ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, stomach(gastric) cancer, testicular cancer, leukemias or lymphomas.